Early Cancer Detection And Enhanced Immunotherapy

ABSTRACT

A method of therapy for a tumor or other pathology by administering a combination of thermotherapy, immunotherapy, and vaccination optionally combined with gene delivery. The combination therapy beneficially treats the tumor and prevents tumor recurrence, either locally or at a different site, by boosting the patient&#39;s immune response both at the time or original therapy and/or for later therapy. With respect to gene delivery, the inventive method may be used in cancer therapy, but is not limited to such use; it will be appreciated that the inventive method may be used for gene delivery in general. The controlled and precise application of thermal energy enhances gene transfer to any cell, whether the cell is a neoplastic cell, a pre-neoplastic cell, or a normal cell.

This patent application claims priority to U.S. Provisional PatentApplication No. 62/569,592, entitled “Cancer Treatment Methods UsingThermotherapy and/or Enhanced Immunotherapy”, filed on Oct. 8, 2017, andto U.S. Provisional Patent Application No. 62/577,485, entitled “CancerTreatment Methods Using Thermotherapy and/or Enhanced Immunotherapy”,filed on Oct. 26, 2017, and is a continuation-in-part of applicationSer. No. 15/143,981, entitled “Early Cancer Detection And EnhancedImmunotherapy”, filed May 2, 2016, which is a continuation-in-part ofapplication Ser. No. 14/976,321, entitled “Method to Visualize VeryEarly Stage Neoplasm or Other Lesions”, filed Dec. 21, 2015, thedisclosure of each of which is hereby incorporated by reference as ifset forth in their entirety herein.

Various factors may lead one to suspect the presence of a smallcancerous or neoplastic tumor in a patient. Such factors include thepatient's genetic history, environmental conditions to which the patientis or has been exposed, the presence of biomarkers in the patient'sblood, or the presence of a lesion on a patient's skin or mucosalsurface. A small neoplasm of 1 to 2 millimeters (mm) in diameter,however, is often not recognized unless and until it produces someclinical symptom.

In a patient having a genetic mutation indicating a predisposition tocancer, prophylactic surgical intervention, such as a bilateralmastectomy performed in a patient having a genetic mutation indicating apredisposition to breast cancer, is seldom performed. Additionally, agenetic predisposition to one type of cancer may not lead to that typeof cancer, e.g. breast cancer, but it may lead to another unsuspectedtype of cancer, e.g. malignant melanoma. Even if the other type ofcancer is suspected, because of the finding of biomarkers in the blood,a small internal lesion may not be seen on radiography, or may not beaccessible by surgery, or the collateral complications may not beacceptable. It may not suffice to just know the biomarker for a tumor,because this information may not indicate whether the tumor is a primarysite or a metastatic site, the tissue of its origin, and/or itslocation. It is appreciated that some treatment techniques such assurgery or radiation may be useful, but only if the tumor is tissuespecific. Radiation and chemotherapy also have their own side effects,and may not destroy the tumor completely. Larger tumors present a muchcomplex problem, e.g., mutations in one area of the tumor are usuallydifferent from mutations in another area of the same tumor.

It is clearly preferable, then, to manage small early neoplasms thathave not progressed to a larger tumor to provide the patient an improvedclinical prognosis.

The invention includes a method of therapy for a non-surgicallyaccessible tumor by administering a combination of thermotherapy andimmunotherapy combined with gene delivery. The combination therapybeneficially treats the tumor and prevents tumor recurrence, eitherlocally or at a different site, by boosting the patient's immuneresponse both at the time or original therapy and/or for later therapyas a “booster” vaccine with or without viral-like particles (VLP) to theoriginal therapy by administering them with checkpoint inhibitors, suchas PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. andanti-inflammatory agents, such as Rock inhibitors, such as Fasudil etc.,Wnt inhibitors, such as niclosamide etc. to enhance cellular immuneresponse of the patient while reducing the inflammatory response andpreventing an auto immune reaction or cytokine storm. In one embodiment,the vaccine with or without VLP is combined with checkpoint inhibitors,such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. andanti-inflammatory agents, Rock inhibitors, such as Fasudil or Botox, orWnt inhibitor, such as niclosamide, ivermectin, etc. can be injectedevery six months or once a year, or is used in the treatment ofrecurrent metastatic disease. In another embodiment the antibody coatednanoparticles are coated with thermosensitive polymers which arereleased at the temperature or 41-42 C under thermotherapy andadministered with checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor 15D11, etc. and Rocks inhibitors, such as Fasudil orBotox, or Wnt inhibitor, such as niclosamide or ivermectin, and anantineoplastic medication depending on the cancer, at lower dose than isnormally recommended, but is made more effective by thermotherapy.

With respect to gene delivery, the inventive method may be used incancer therapy, but is not limited to such use; it will be appreciatedthat the inventive method may be used for gene delivery in general. Forexample, the inventive method facilitates cellular gene uptake bycurrent methods that lack a thermal energy component, such aselectroporation, quantum dot delivery, etc. The controlled and preciseapplication of thermal energy enhances gene and medication transfer toany cell, whether the cell is a neoplastic cell, a pre-neoplastic cell,or a normal cell.

The inventive method provides in vitro and in vivo precisionimmunotherapy to decrease or eradicate a malignant neoplasm at an earlystage of the disease. This method provides a vaccination effectperiodically to prevent at least the same kind of cancer or to treatrecurrences.

One embodiment is a method for evaluating treatment outcome in a patienthaving a genetic predisposition for a malignant neoplasm before clinicalmanifestation of the neoplasm can be seen radiographically. The methodpermits visualization of any tumor, whether located externally on apatient's body or located internally in the body, and as small as 2 mmin diameter, producing a biomarker, either a biomarker specific for thetumor or a general biomarker.

In general, a biomarker indicates a disease process. As subsequentlydescribed, a biomarker can be a protein, antigen, enzyme, hormone,carbohydrate, toxin, DNA, an organism such as bacteria, tumor cell,exosome, or indirectly an antibody, present in a liquid biopsy specimen.It can be produced by the plasma cells, against a tumor antigen, etc.

The method uses antibodies conjugated with nanoparticles which includebut are not limited to quantum dots, with the conjugated formcollectively termed functionalized nanoparticles, that are heated underspecified conditions to produce a photoacoustic or thermoacoustic signalthat is then recorded and visualized to locate the tumor to which thenanoparticles are attached. Nanoparticles may be used for qualitativeand quantitative assessment of an analyte in the blood or other tissueusing photoacoustic/thermoacoustic technology, U.S. Pat. No. 8,554,296.As previously stated, as used herein, unless specifically statedotherwise, nanoparticles include but are not limited to quantum dots.

Early stage small neoplastic cells produce biomarkers that are eitherspecific to the tumor cells or that represent the body's response to thetumor as an antibody. The biomarkers can be proteomic, genetic,epigenetic or glycomic biomolecules. These biomolecules can berecognized in the patient's tissue samples or in the blood or fluids.Their existence can be demonstrated thus far chemically using, e.g.,immunoassay or PCR methods. Quantitation of these biomarkers is alsoimportant to determine disease progression and prognosis of the disease.

Biomarkers for many diseases are found in the blood. As subsequentlydisclosed, biomarkers detected in a liquid biopsy sample are used togenerate antibodies against them using known methods in the art. Theanti-tumor antibodies are used to coat nanoparticles in the inventivemethod, where a lesion can be imaged regardless of the lesion size orlocation in the body. The method is not limited to tumor detectionand/or therapy. As only one example, detecting an antibody againstanti-beta-amyloid protein present in Alzheimer's disease in a liquidbiopsy specimen, the method renders the plaque visible with thenanoparticles and accessible to the inventive treatment. As anotherexample, the method can also be used to detect and/or treat inflammatoryprocesses, etc.

The inventive method is applicable to any processes or diseases thatproduce a biomarker detectable in a liquid biopsy specimen. It isapplicable to a lesion including an abscess, an ulcer, a tumor eitherbenign or malignant, an ischemic area of a stroke and/or an area of thebrain affected by a stroke whether visible or microscopically.

Well over a thousand proteins are differentially expressed in humancancers and thus may serve as biomarkers. Such proteins play a role incancer-related processes such as angiogenesis, apoptosis, celldifferentiation, cell signaling, hematopoiesis, hormonal control, immunereactions, etc. Exemplary biomarkers include, but are not limited to,CEA for both malignant pleural effusion and peritoneal cancerdissemination; HER-2/neu for stage IV breast cancer; bladder tumorantigen for urothelial cell carcinoma; thyroglobulin for thyroid cancermetastasis; α-fetoprotein for hepatocellular carcinoma; PSA for prostatecancer; CA 125 for non-small cell lung cancer; CA 19.9 for pancreaticcancer; CA 15.3 for breast cancer; the combination of leptin, prolactin,osteopontin, and IGF-II for ovarian cancer; the combination of CD98,fascin, sPIgR, and 14-3-3 eta for lung cancer; troponin I for myocardialinfarction, and B-type natriuretic peptide for congestive heart failure.While the previous nine proteins are the only approved markers forcancer testing to date, they are but a small fraction of the totalnumber of available biomarkers, and their sensitivity and specific vary.

Other common biomarkers include the estrogen receptor/progesteronereceptor (ER/PR), HER-2/neu, and ESFR for breast cancer, andTIMP-1-associated with serum HER2-positive breast cancer; KRAS andUGT1A1 for colorectal cancer; HER-2/neu for gastric cancer, c-KIT, CD20antigen, CD30, and FIP1L1-PDGRF alpha, and PDGFR for GIST; PhiladelphiaChromosome (BCR/ABL)/PML/RAR alpha and TPMT/UGT1A1/ALK EGFR forleukemia/lymphoma; KRAS/EGFR for lung cancer, and BRAF and S100 formelanoma.

Other examples of biomarkers include tumor suppressors that are lost incancers, such as BRCA1, BRCA2; RNA such as mRNA, microRNA; proteinsfound in body fluids or tissue such as prostate specific antigen andCA-125; gene and protein based biomarkers; and nonspecific biomarkerssuch as glycosaminoglycans in body fluids; alkaline phosphatase andurinary hydroxyproline in skeletal involvement; hyaluronic acidexcretion and urinary hydroxyproline in bone disease, and combinationsthereof.

In malignancies, the biomarkers may be released into the circulationeither prior to or after the tumor has grown sufficiently to becomemetastatic. Small tumors (less than about 2 mm) seldom have any clinicalmanifestations, however even such small tumors can release chemicaland/or biomarkers into the circulation.

The existence of biomarkers in the circulation has been known, but hasnot met the threshold for locating tumor cells that could not be imagedradiographically or by ultrasound as long as the tumors wereasymptomatic. Available imaging methods such as x-ray, magneticresonance imaging (MRI), functional MRI, computed tomography (CT) scans,CT ultrasound, etc. may not permit visualization of lesions smaller thanabout 3 mm in diameter. This has been the case for most malignanttumors, or when a malignant tumor is created from a benign precursorlesion such as nevus, breast unspecific cyst or unspecific scar,prostate tumors along with benign prostate hypertrophy, or uterus cancerinside the uterus fibroma, melanoma inside a skin nevus or choroidalnevus under the retina in the eye or in a seborrheic keratosis, etc.Moreover, it is often difficult to follow a cancerous tumor which hasbeen irradiated but may still harbor malignant cells, and that can startgrowing with time and metastasize before it shows a local growth that isdetected by conventional imaging or other methods.

The diagnosis of a malignant tumor may be extremely difficult, even whena tumor is visible clinically or radiologically, e.g. a uterus fibromathat may have some malignant transformation. Moreover, a diagnosis alsoaffects the decision whether or not and also how to remove the tumor. Asone example, accessing the uterus through a small incision, and removingthe tumor piece by piece using an endoscope and a cutting probe, has afast post-operative recovery. Such a method is in contrast to completelyremoving the uterus with the tumor intact out of caution that the tumormay harbor neoplastic cells, but using a large incision withsignificantly higher operative risks and post-operative complicationprobabilities. Another, more problematic example, is the decision for awoman having genetic disposition to breast cancer without any physicalor radiological manifestation. The woman must endure the stress and fearnot knowing if or when she may develop breast cancer, and must considerprophylactic removal of both breasts. As another example, a personaldecision whether or not to undergo radiation therapy when a nevus isdiscovered under the retina, and biopsy results that often do notprovide definitive information because of the diversity of the cells inthe entire area of the tumor.

When the tumor site is unknown, locating a biomarker in the circulationmay be akin to finding a needle in a hay stack. For any particular tumoror cancer, not all biomarkers are even known. Similarly, finding a microDNA in the circulation may not provide an answer when the tumor iseither invisible or has already metastasized. An example of this occursin patients with uveal melanomas, having a mortality rate of about 50%,even if the tumors undergoes radiation, at the time the ophthalmologistdiscovers the tumor. This points to the fact that a malignant tumor canmetastasize very early, at times even when the size of the tumor isabout 2 mm in diameter which is equal to about one million cells. Ingeneral, these lesions do not have any symptoms.

The inventive method makes it possible to evaluate a patient withgenetic predisposition of a malignant neoplasm before its clinicalmanifestation can be seen radiographically.

In one embodiment, the presence of one or more biomarkers is evaluatedin any body fluid or organ. Exemplary bodily fluids include, but are notlimited to, urine, blood, cerebrospinal fluid (CSF), eye cavity fluid,tear film, sputum, fluid obtained from the trachea, bronchi, abdominalcavity, vagina, uterus etc. The biomarkers are analyzed in vitro bymethods known in the art, e.g., immunoassays including enzyme-linkedimmunoassay (ELISA), Western blots, fluorescence in situ hybridization(FISH), polymerase chain reaction (PCR), etc. The biomarkers are thenconjugated with functionalized antibody coated nanoparticles and/orquantum dots, as known in the art.

In one embodiment one obtains a liquid biopsy sample. Such a sample maybe obtained from, e.g., blood, urine, cerebrospinal fluid (CFS), aqueousor vitreous or abdominal cavity fluid, lymph node fluid, bladder fluid,milk duct fluid, sputum, gastric fluid, bile duct fluid, sinus fluid,etc. The patient may or may not have any clinical symptom. The patientmay or may not have history of a family disposition for tumors in and/orcancer of the breast, brain, lung, prostate, ovary, pancreas, etc., or agenetic abnormality leading to progression in diseases such as, e.g.,Alzheimer's, Parkinson's, post traumatic brain syndrome, brain tumor,other neurological disease, age related macular degeneration, aninfectious disease, an immune response, etc. The method evaluates thecomponents of the sample for cell free nucleic acid-based biomarkersincluding but not limited to micoRNA and microDNA; protein-basedbiomarkers, extracellular vesicle (EV)-based biomarkers that arecontained within exosomes, extracellular vesicles, or microvesicles, andcirculating tumor cell (CTC)-based biomarkers. The method usesmethodologies such as next generation sequencing (NGS) or recombinantaffinity reagents fabricated into nanostructures such as carbonnanotubes, nanowires, quantum dots, or gold nanoshells, to enhance theirdetection with the use of, e.g., surface-enhanced Raman scattering(SERS), as known in the art.

For example, if a known tumor exists and there is a known biomarker forthe tumor, one may have or prepare an antibody against the tumor to beused in both imaging and therapy. Large tumors with symptoms can beimaged, but before the inventive method, there was a problem when abiomarker was present in a liquid biopsy specimen but the tumor wasinvisible, e.g., an early stage of a tumor, and there was no symptomaticor radiographic evidence of the tumor.

Detecting a tumor biomarker, typically a protein or a glycoprotein, in aliquid biopsy specimen is facilitated by the inventive method. Oncedetected, an antibody against that tumor biomarker can be prepared. Theantitumor biomarker antibody is used to located the tumor. Antibodyproduction is a well-known method in the art, and it will be appreciatedthat the antibody against either or both of the tumor biomarker and thetumor cell may be recombinant, monoclonal, polyclonal, or an aptamer.The prepared antitumor cell antibodies are conjugated with nanoparticlesand administered to a patient, where they target the tumor cells and canbe detected and/or treated. Detection is by photoacoustic/thermoacousticimaging technology. Treatment is at least by one of thermal energy. Thephotoacoustic detection and thermal treatment is described herein.

In one embodiment, any specific tumor related biomarker may be used. Oneexample uses trastuzumab or herceptin, a recombinant monoclonalantibody, against the oncogene HER-2, previously mentioned, which is amember of the human epidermal growth factor receptor (HER/EGFR/ERBB)family. Other examples of known monoclonal antibodies or biologicsinclude, but are not limited to, rituximab, cetuximab, racotunomab,obinotuzumab, pertuzumab, belaniatumomab, bevacizumab, nivolumab,ofatumumab, botezomib, daratumumab, ipilumumab, pembrolizumab, anddaratumumab.

In one embodiment, in the absence of a specific biomarker, antibodiesagainst biomarkers that are shared by a number of the tumors may beused. Such biomarkers include glycosaminoglycan, which is specific for agroup of cancers such as bladder, gastrointestinal, glioblastoma, etc.Antibodies against such biomarkers are then conjugated withnanoparticles, termed functionalized nanoparticles. The term“functionalized” indicates nanoparticles that have been coated to renderthem soluble, biocompatible, and/or targeted by conjugating them with abiomolecule such as an antibody.

In one embodiment, the pluralities of nanoparticles may be one or moreof the following compounds or contain one or more of the followingcomponents: quantum dots, nanowires, nanotubes, nanoshells, nanocages,perovskites, nanoparticles that are magnetic such as iron or iron oxide,paramagnetic, or nanoparticles that are non-magnetic such as gold,gold-silica, gold-iron, silica coated gold nanospheres and nanorods,ferritic, quartz, graphene, carbon, zinc oxide, piezoelectric, etc. Anyof these nanoparticles, alone or in combination, may be conjugated orotherwise associated with the biomarkers' antibodies, using methodsknown in the art.

In another embodiment, self-assembling bio/nano hybrid materialconsisting of two constituents at the nanometer or molecular levelcomposed of inorganic and organic compounds, having amphiphiliccharacteristics, i.e., hydrophilic and lipophilic components ormicelles, which may be radioactive (e.g., Cu⁶⁴) or radioactive (e.g.,tin) are prepared with biocompatible coatings and administered in thebody for both therapy and imaging.

In one embodiment, the functionalized nanoparticles travel in the bodyand attach to receptors of desired cells, e.g., tumors, Alzheimer'splaque, drusen of the retina, etc. These nanoparticles are imaged byapplying external thermal energy and/or by applying an electromagneticradiation, microwave, radiofrequency waves or a reversible oralternating magnetic field. The thermal energy causes the nanoparticlesto expand, producing an ultrasound wave in the tissue known asphotoacoustic or thermoacoustic sound. The ultrasound wave can bedetected by an ultrasonic receiver which is imaged in two to threedimensional formats as a tomogram. In another embodiment the plaques inAlzheimer's disease, and the drusen in age related macular degeneration,are rendered visible using silica coated nanoparticles<2 nm in diameteradministered with turmeric, glycosaminoglycan, amyloid antibody, orpercolan, etc. and are quantified. In another embodiment, thenanoparticles are conjugated with thermosensitive polymers, such aschitosan, polylactic poly glycolic acid, acrylic derivatives,polycaprolactone, and are conjugated with are conjugated withantibodies, medications, sterols, antibiotics, antifungals,antibacterials, antiproliferative agents, medications interfering withthe normal cell signaling processes, with stimulatory or inhibitoryaction such as Wnt inhibitors ivernectin or Rho kinase inhibitors knownas Rock inhibitors (e.g., fasudil or Botox) or dyes, etc. that can bereleased from silica coated gold nanoparticles when coated withthermosensitive polymers, e.g., chitosan coated nanoparticles heated to40° C.-42° C., to treat various diseases including bacteria, fungi,parasites, plaque, drusen, inflammation, tumors, etc. In anotherembodiment, the plaques and drusen can be quantified by imaging usinglight, MRI, photoacoustic/thermoacoustic technology imaging, etc.

In another embodiment, the functionalized anti-biomarker-conjugatednanoparticle, ranges in size from 1 nm to 900 nm. In another embodiment,the functionalized biomarker ranges in size from 1 nm to 8 nm, chosen toenhance their elimination through the kidney for facilitated clearance.

In one embodiment, the nanoparticles are rendered magnetic by coatingwith a thin film of iron oxide prior to their conjugation withbiomarkers' antibodies.

In one embodiment, the nanoparticles are rendered more biocompatible bycoating with a compound, including but not limited to the following:(poly)ethylene glycol, cell penetrating peptide (CPP), activating CPP(ACPP), biotin, streptavidin, etc., as known in the art, prior to theirinjection in the body.

Thermal energy in the form of electromagnetic radiation, ultrasound, oran alternating magnetic field is applied, under the control of aphotoacoustic/thermoacoustic imaging system, to the organ suspected ofpotentially harboring an as yet invisible neoplasm. The thermal energyapplied increases preferentially the temperature of the exposednanoparticle, and creates (e.g. using a laser light) a photoacousticsound from the superficial lesions and or using focused ultrasound,microwave, RF, or an alternating magnetic field to produce athermoacoustic sound from deep in the tissue located lesions, and toimage or create a tomogram of the accumulated heatednanoparticles/tumor. This image or tomogram represents a suspectedneoplasm in that organ, and is compared to an image taken without thethermal application radiographically.

In one embodiment, one administers functionalized antibody-coatednanoparticles that, once attached to tumor cells, become visible with aphotoacoustic/thermoacoustic imaging unit that corroborates with animage obtained or not seen with other technology such as ultrasound,MRI, PET, CT scan, etc. In one embodiment, the images obtained withother instruments are either overlapped using a processor or are takensimultaneously during photoacoustic/thermoacoustic imaging. In oneembodiment, after administration of the antibody-coated nanoparticle, anMRI image is overlapped with the photoacoustic image and compared by aprocessor to verify the changes in the imaged area.

In one embodiment, the nanoparticles are incorporated in liposomes. Inthis embodiment, they may contain medications or a dye that, uponattainment of a specific tumor temperature, are released. The type ofmedication is not limited, and can include anti-bacterial, anti-viral,anti-fungal, antineoplastic, anti-inflammatory such as acetyl cycline,anti-beta-amyloid protein, other antibodies, non-steroidalanti-inflammatory drugs, Rock inhibitors, Botox, Wnt inhibitorsniclosamide, ivernectin, preventing inflammation or tumor growth,checkpoint inhibitors, immune stimulating agents, VLPs, anti-VEGFagents, anti-aggregation agents such as sterols, etc.

In another embodiment, antibody-coated nanoparticles conjugated withthermosensitive polymers such as chitosan, carrying any medicationincluding but not limited to sterol, squalamine, lanosterol, isadministered to a patient having a neurologic pathology such asAlzheimer's disease, Parkinson's disease, or age related retinal drusen,etc. In this embodiment, administration is either intravenous or localin the cerebrospinal fluid or vitreous cavity, respectively, or atanother local site. After controllably increasing the temperature of thefunctionalized nanoparticle to between 40° C.-43° C. by increased energydelivery through a delivery source, under the control of thephotoacoustic imaging system and a processor, the temperature-sensitivecoating polymers such as chitosan melts and release medications specificto the pathology. For example, a medication to dissolve amyloid plaqueswould be administered to a patient with Alzheimer's disease; amedication to remove retinal drusen would be administered to a patientwith age related retinal disease, etc.

In one embodiment, the functionalized nanoparticle, e.g., a nanoshell,nanocage, etc., is combined with biodendrimers that are conjugated withbiomarkers and monoclonal antibodies and/or genes, e.g., siRNA, mRNA,etc., for simultaneous visualization and therapy.

In another embodiment, after thermal imaging one increases thetemperature of the functionalized nanoparticles. This is achieved byincreased energy delivered by a thermal delivery source under thecontrol of the photoacoustic/thermoacoustic imaging system connected toa processor. The energy delivery unit increases the temperature of thefunctionalized nanoparticles to 41° C.-43° C. to melt thetemperature-sensitive coating polymers such as chitosan, liposomes, andrelease anticancer medications, Rock inhibitors (e.g., fasudil,exoenzyme or Y27632, Botox etc.), Wnt inhibitors (e.g., niclosamide,etc.) or inhibitory genes, siRNA, miRNA, or checkpoint inhibitors, orintroduce missing genes, or add any other genes for gene editing fromthe thermosensitive coating of the nanoparticles along with a CRISPRcomplex to modify the genetic composition of the tumor cells, etc. Inanother embodiment, the temperature of the functionalized nanoparticlesis increased, by the thermal delivery unit via a processor under thecontrol of the photoacoustic/thermoacoustic imaging unit to image thetemperature and control it to 45° C.-47° C., to 47° C., or to 50° C. tokill the suspected tumor to which the antibody-coated nanoparticles areattached and release tumor antigens in the circulation to attract andenhance a cellular immune response to a tumor.

In one embodiment, one synthetizes hybrid, very small (1 nm-8 nm) goldsilica nanoparticles that have a dual function, the nanoparticlesantibody coated for imaging, and having photovoltaic and magneticproperties, to release one or more gene(s) or medication(s) at certaintemperatures, creating a photoacoustic/thermoacoustic signal afterheating for imaging in the body or by laser or light stimulation in theeye for simultaneous imaging and therapy.

In one embodiment, using antibody coated quantum dots and light of aspecific wavelength that is absorbed by the quantum dot and emits lightof a different wavelength, one can render the moving tumor cells andextracellular vesicle visible attached to the quantum dots visible inthe retinal or choroidal vessels, or vessels and tumors of the skin, ortumors located beneath the skin and their feeding vessels, by lightabsorbed by the quantum dots circulating in the vessels, as is done influorescence angiography with appropriate filters and camera.

In another embodiment, a gold quantum dot in a mesoporous silica shellor cage is coated with an antibody or a biomarker to any cell, e.g.,neuronal or tumor cells, retinal drusen, Alzheimer plaques, etc. fordelivering medication or gene to an organ, e.g., retina or brain.

In another embodiment, the extent of plaque or drusen, as an indicatorof disease progression in the brain or eye, respectively, can beevaluated by conjugating nanoparticles with antibodies toglycosaminoglycan, heparin sulfate, glycosaminoglycan, and/or heparinsulfate proteoglycan, and injecting the composition into the circulationof the body or locally to adhere to plaques or drusen for diagnosis,quantitation, and/or therapy with antibodies and medication.

In another embodiment, the pluralities of antibody coated nanoparticlesare used for simultaneous imaging and thermotherapy of very smalltumors. The nanoparticles are heated to a temperature ranging from 41°C.-43° C., releasing anti-cancer medication, such as rock inhibitors,such as Botox, exoenzyme or Y27632, or Wnt inhibitors (e.g., niclosamideor ivermectin) precisely at the desired location, along with inhibitorysiRNA, or modify a gene using the CRISPR/cas9 system or another CRISPRsystem, additionally releasing checkpoint inhibitors such as CTLA-4 orPD-1 or Jagged 1 along with tumoricidal vectors, etc.

In one embodiment, the pluralities of antibody coated nanoparticles arerendered radioactive by coating with alpha or beta radiators that areantibody specific or nonspecific biomarkers of the tumor. Thenanoparticles can also be coated with heat sensitive polymers, includingbut not limited to chitosan, PEG, poly amino esters, etc.

In one embodiment, checkpoint inhibitors defined as immune systemcomponents that act as co-stimulatory or co-inhibitory molecules arereleased from the pluralities of antibody coated nanoparticles fromthermosensitive coating of the nanoparticles at temperature of 41 to 43degrees C. along with poisons such as bee or snake venom, or other toxicagents that damage tumor cell membranes, or genes that inhibit tumorgrowth, siRNA, siDNA, mi RNA, mDNA along with the CRISPR/cas 9 complexor variations of these may be used.

In one embodiment, the pluralities of antibody coated nanoparticles arecoated with a specific or a nonspecific biomarker such asglycosaminoglycan and injected into the circulation, into a body fluidsuch as the lymphatic system or cerebrospinal fluid (CSF), or inside abody cavity. Examples of injection sites include, but are not limitedto, eye, sinuses, abdominal cavity, bladder, uterus, etc. Thenanoparticles may also be injected into the breast ducts, e.g., throughthe nipple, inside the brain, into the prostate or other organ, or mayeven be applied topically. The injected nanoparticles circulate and seekcells bearing a receptor to their antibody, or perhaps cells withspecific receptors or biomolecules, and readily attach within minutes orhours.

In one embodiment, specific or non-specific biomarkers' antibodies areconjugated with nanoparticles and injected either into circulation orlocally into a body cavity. The nanoparticles travel and seek cellsbearing specific receptors or biomolecules, and attach within a fewhours. The patient's body or organ is then scanned, with the thermalenergy producing radiation or an alternating or reversible magneticfield, microwave radiation, a laser, radiofrequency (RF) waves orfocused ultrasound to heat the nanoparticles. Usingphotoacoustic/thermoacoustic technology, the sound wave generated by thethermal expansion of the nanoparticle induced by absorption of thethermal energy is recorded. The sound wave signals may originate fromany part of the body, or from a specific organ.

In one embodiment, an alternating magnetic field produces heat inmagnetic nanoparticles as a result of rapid circular or semicircularmotion of the magnetic or paramagnetic nanoparticles heating them. Thepatient's body is scanned within the reversible magnetic field, and thephotoacoustic sound is recorded as a temperature profile of the site ofthe nanoparticle/cell membrane imaged and location of the lesion isverified.

In another embodiment, other sources of thermal energy are used. Suchsources include, but are not limited to, electromagnetic radiation,visible light, invisible light, infrared radiation, microwaves, orradiofrequency waves, focused ultrasound (FUS), etc. The nanoparticlesare heated from body temperature of 37° C. to 40° C. or 43° C., or ifneeded to 45° C. At the desired temperature, e.g., 41° C.-43° C., theheat sensitive coating of the nanoparticle melts, releasing its cargoof, e.g., medication, gene, etc., thus facilitating or enhancing passageof these compounds through the membrane of the neoplastic cells.

In another embodiment, use of a photoacoustic technology unit controlsthe thermal delivery unit and the thermal energy delivered to thenanoparticles to maintain or reach a predetermined temperature for adesired time.

In one embodiment, the temperatures rise of the nanoparticles expandsthem, producing a photoacoustic or thermoacoustic sound wave. This soundwave is recorded by one or multiple ultrasonic receivers located on thepatient's skin. The signal can be obtained from any part of the body, orfrom a specific organ, since the signal travels through the body as awave. The signal or sound pulse is converted to an electric pulse in thereceiver, then using a processor, is amplified and imaged on a monitor.A processor produces a two- or three-dimension image of the lesion,localizing the location of the sound and indicating the size of a lesionand its temperature by the amplitude of the sound pulse.

In one embodiment, photoacoustic imaging is used for a very early stagediagnosis of cancerous lesion that are less than 2 mm in diameter, whichare radiographically invisible without knowing their exact location inthe body.

In one embodiment using photoacoustic technology and a specific ornon-specific tumor biomarker, a very small lesion (<2 mm in diameter) isimaged in the body when the tumor has not caused any clinical symptom.The inventive method thus is used to differentiate a malignant lesionfrom a benign lesion, even if the cancerous lesion is inside a beginlesion. It is noteworthy that biopsy of these very small tumors, evenwhen the lesion is visible, e.g., on skin or under the retina, may notyield malignant cells if the biopsy is performed on a part of the lesionthat contains benign cells. With tumors in the brain, it is most oftenthe case that the tumors will not be noted absent a neurologicalsymptom.

In one embodiment, the inventive method is used with specific biomarkersof a tumor such as breast cancer, prostate cancer, glioma, pancreaticmalignancies, along with nonspecific biomarkers. The location and sizeof a malignant tumor in any organ is imaged in a patient with a geneticpropensity to develop a tumor. The thermal energy may also be applied,if desired, to treat the lesion simultaneously with providing thephotoacoustic effect. Subsequent evaluation of the level of thesebiomarkers in the blood indicate if the lesion was damaged or eliminatedby the thermal energy increasing the biomarkers in the blood, includinguse of medicaments/dye released from the thermosensitive nanoparticlecoating and/or other treatment agents delivered by the method as cargoin the nanoparticles.

In one embodiment, a combination of biomarkers can be used in an earlystage. For example, specific or nonspecific bio-markers such asglycosaminoglycans can be used in imaging a malignant lesion usingantibody-coated nanoparticles to photoacoustically image the presence ofa very small early stage tumor anywhere in the body.

In another embodiment, the inventive method is employed to determineresidual tumor cells that may have left at the site of a tumor resectionor elsewhere in the body, and to treat or eliminate the residual tumorcells.

In another embodiment, the functionalized nanoparticles are conjugatedwith one of the recombinant, monoclonal, or polyclonal antibodies oraptamers known in the art and administered along with either one or moretoxin(s) or antibodies, along with a medication that is provided at amuch lower dose systemically to kill the already compromised tumorcells. Monoclonal antibodies that may be used include, but are notlimited to, those shown in Table 1, e.g., rituximab, obinuzumab,oftumumab, etc.

TABLE 1 Name Trade name Type Source Target Use 3F8 mab mouse GD2neuroblastoma 8H9 mab mouse B7-H3 neuroblastoma, sarcoma, metastaticbrain cancers Abagovomab mab mouse CA-125 (imitation) ovarian cancerAbciximab ReoPro Fab chimeric CD41 (integrin alpha- platelet aggregationIIb) inhibitor Abituzumab mab humanized CD51 cancer Abrilumab mab humanintegrin α4β7 inflammatory bowel disease, ulcerative colitis, Crohn'sdisease Actoxumab mab human Clostridium difficile Clostridium difficileinfection Adalimumab Humira mab human TNF-α Rheumatoid arthritis,Crohn's Disease, Plaque Psoriasis, Psoriatic Arthritis, AnkylosingSpondylitis, Juvenile Idiopathic Arthritis, Hemolytic disease of thenewborn Adecatumumab mab human EpCAM prostate and breast cancerAducanumab mab human beta-amyloid Alzheimer's disease Afelimomab F(ab′)₂mouse TNF-α sepsis Afutuzumab mab humanized CD20 lymphoma Alacizumabpegol F(ab′)₂ humanized VEGFR2 cancer ALD518 ? humanized IL-6 rheumatoidarthritis Alemtuzumab Campath, mab humanized CD52 Multiple sclerosisMabCampath Alirocumab mab human NARP-1 hypercholesterolemia Altumomabpentetate Hybri-ceaker mab mouse CEA colorectal cancer (diagnosis)Amatuximab mab chimeric mesothelin cancer Anatumomab mafenatox Fab mouseTAG-72 non-small cell lung carcinoma Anetumab ravtansine mab human MSLNcancer Anifrolumab mab human interferon α/β receptor systemic lupuserythematosus Anrukinzumab (= IMA- mab humanized IL-13 ? 638⁾ Apolizumabmab humanized HLA-DR ? hematological cancers Arcitumomab CEA-Scan Fab′mouse CEA gastrointestinal cancers (diagnosis) Ascrinvacumab mab humanactivin receptor-like cancer kinase 1 Aselizumab mab humanizedL-selectin (CD62L) severely injured patients Atezolizumab mab humanizedCD274 cancer Atinumab mab human RTN4 ? Atlizumab (= Actemra, mabhumanized IL-6 receptor rheumatoid arthritis tocilizumab) RoActemraAtorolimumab mab human Rhesus factor hemolytic disease of thenewborn[citation needed] Bapineuzumab mab humanized beta amyloidAlzheimer's disease Basiliximab Simulect mab chimeric CD25 (α chain ofIL-2 prevention of organ receptor) transplant rejections Bavituximab mabchimeric phosphatidylserine cancer, viral infections BectumomabLymphoScan Fab′ mouse CD22 non-Hodgkin's lymphoma (detection) Begelomabmab mouse DPP4 ? Belimumab Benlysta, mab human BAFF non-Hodgkin lymphomaLymphoStat-B etc. Benralizumab mab humanized CD125 asthma Bertilimumabmab human CCL11 (eotaxin-1) severe allergic disorders BesilesomabScintimun mab mouse CEA-related antigen inflammatory lesions andmetastases (detection) Bevacizumab Avastin mab humanized VEGF-Ametastatic cancer, retinopathy of prematurity Bezlotoxumab mab humanClostridium difficile Clostridium difficile infection BiciromabFibriScint Fab′ mouse fibrin II, beta chain thromboembolism (diagnosis)Bimagrumab mab human ACVR2B myostatin inhibitor Bimekizumab mabhumanized IL17A and IL17F ? Bivatuzumab mertansine mab humanized CD44 v6squamous cell carcinoma Blinatumomab BiTE mouse CD19 cancer Blosozumabmab humanized SOST osteoporosis Bococizumab mab humanized neuralapoptosis- dyslipidemia regulated proteinase 1 Brentuximab vedotin mabchimeric CD30 (TNFRSF8) hematologic cancers Briakinumab mab human IL-12,IL-23 psoriasis, rheumatoid arthritis, inflammatory bowel diseases,multiple sclerosis Brodalumab mab human IL-17 inflammatory diseasesBrolucizumab mab humanized VEGFA ? Brontictuzumab mab Notch 1 cancerCanakinumab Ilaris mab human IL-1? rheumatoid arthritis Cantuzumabmertansine mab humanized mucin CanAg colorectal cancer etc. Cantuzumabravtansine mab humanized MUC1 cancers Caplacizumab mab humanized VWFthrombotic thrombocytopenic purpura, thrombosis Capromab pendetideProstascint mab mouse prostatic carcinoma prostate cancer cells(detection) Carlumab mab human MCP-1 oncology/immune indicationsCatumaxomab Removab 3funct rat/mouse hybrid EpCAM, CD3 ovarian cancer,malignant ascites, gastric cancer cBR96-doxorubicin mab humanizedLewis-Y antigen cancer immunoconjugate Cedelizumab mab humanized CD4prevention of organ transplant rejections, treatment of autoimmunediseases Certolizumab pegol Cimzia Fab humanized TNF-α Crohn's diseaseCetuximab Erbitux mab chimeric EGFR metastatic colorectal cancer andhead and neck cancer Ch.14.18 mab chimeric ??? neuroblastoma Citatuzumabbogatox Fab humanized EpCAM ovarian cancer and other solid tumorsCixutumumab mab human IGF-1 receptor solid tumors Clazakizumab mabhumanized Oryctolagus cuniculus rheumatoid arthritis Clenoliximab mabchimeric CD4 rheumatoid arthritis Clivatuzumab tetraxetan hPAM4-Cide mabhumanized MUC1 pancreatic cancer Codrituzumab mab humanized glypican 3cancer Coltuximab ravtansine mab chimeric CD19 cancer Conatumumab mabhuman TRAIL-R2 cancer Concizumab mab humanized TFPI bleeding Crenezumabmab humanized 1-40-β-amyloid Alzheimer's disease CR6261 mab humanInfluenza A infectious hemagglutinin disease/influenza A Dacetuzumab mabhumanized CD40 hematologic cancers Daclizumab Zenapax mab humanized CD25(α chain of IL-2 prevention of organ receptor) transplant rejectionsDalotuzumab^([39]) mab humanized insulin-like growth cancer etc. factorI receptor Dapirolizumab pegol mab humanized CD40 ligand ? Daratumumabmab human CD38 (cyclic ADP cancer ribose hydrolase) Dectrekumab mabhuman IL-13 ? Demcizumab mab humanized DLL4 cancer Denintuzumabmafodotin mab humanized CD19 cancer Denosumab Prolia mab human RANKLosteoporosis, bone metastases etc. Derlotuximab biotin mab chimerichistone complex recurrent glioblastoma multiforme Detumomab mab mouseB-lymphoma cell lymphoma Dinutuximab mab chimeric ganglioside GD2neuroblastoma Diridavumab mab human hemagglutinin influenza A Dorlimomabaritox F(ab′)₂ mouse ? ? Drozitumab mab human DR5 cancer etc.Duligotumab mab human HER3 ? Dupilumab mab human IL4 atopic diseasesDurvalumab mab human CD274 cancer Dusigitumab mab human ILGF2 cancerEcromeximab mab chimeric GD3 ganglioside malignant melanoma EculizumabSoliris mab humanized C5 paroxysmal nocturnal hemoglobinuria Edobacomabmab mouse endotoxin sepsis caused by Gram- negative bacteria EdrecolomabPanorex mab mouse EpCAM colorectal carcinoma Efalizumab Raptiva mabhumanized LFA-1 (CD11a) psoriasis (blocks T-cell migration) EfungumabMycograb scFv human Hsp90 invasive Candida infection Eldelumab mab humaninterferon gamma- Crohn's disease, induced protein ulcerative colitisElgemtumab mab human ERBB3 cancer Elotuzumab mab humanized SLAMF7multiple myeloma Elsilimomab mab mouse IL-6 ? Emactuzumab mab humanizedCSF1R cancer Emibetuzumab mab humanized HHGFR cancer Enavatuzumab mabhumanized TWEAK receptor cancer etc. Enfortumab vedotin mab humanAGS-22M6 cancer expressing Nectin-4 Enlimomab pegol mab mouse ICAM-1(CD54) ? Enoblituzumab mab humanized B7-H3 cancer Enokizumab mabhumanized IL9 asthma Enoticumab mab human DLL4 ? Ensituximab mabchimeric 5AC cancer Epitumomab cituxetan mab mouse episialin ?Epratuzumab mab humanized CD22 cancer, SLE Erlizumab F(ab′)₂ humanizedITGB2 (CD18) heart attack, stroke, traumatic shock Ertumaxomab Rexomun3funct rat/mouse hybrid HER2/neu, CD3 breast cancer etc. EtaracizumabAbegrin mab humanized integrin αvβ3 melanoma, prostate cancer, ovariancancer etc. Etrolizumab mab humanized integrin α7 β7 inflammatory boweldisease Evinacumab mab human angiopoietin 3 dyslipidemia Evolocumab mabhuman PCSK9 hypercholesterolemia Exbivirumab mab human hepatitis Bsurface hepatitis B antigen Fanolesomab NeutroSpec mab mouse CD15appendicitis (diagnosis) Faralimomab mab mouse interferon receptor ?Farletuzumab mab humanized folate receptor 1 ovarian cancer Fasinumabmab human HNGF acute sciatic pain FBTA05 Lymphomun 3funct rat/mousehybrid CD20 chronic lymphocytic leukaemia Felvizumab mab humanizedrespiratory syncytial respiratory syncytial virus virus infectionFezakinumab mab human IL-22 rheumatoid arthritis, psoriasis Ficlatuzumabmab humanized HGF cancer etc. Figitumumab mab human IGF-1 receptoradrenocortical carcinoma, non-small cell lung carcinoma etc. Firivumabmab human influenza A virus ? hemagglutinin Flanvotumab mab humanTYRP1(glycoprotein melanoma 75) Fletikumab mab human IL 20 rheumatoidarthritis Fontolizumab HuZAF mab humanized IFN-γ Crohn's disease etc.Foralumab mab human CD3 epsilon ? Foravirumab mab human rabies virusrabies (prophylaxis) glycoprotein Fresolimumab mab human TGF-βidiopathic pulmonary fibrosis, focal segmental glomerulosclerosis,cancer Fulranumab mab human NGF pain Futuximab mab chimeric EGFR ?Galiximab mab chimeric CD80 B-cell lymphoma Ganitumab mab human IGF-Icancer Gantenerumab mab human beta amyloid Alzheimer's diseaseGavilimomab mab mouse CD147 (basigin) graft versus host diseaseGemtuzumab Mylotarg mab humanized CD33 acute myelogenous ozogamicinleukemia Gevokizumab mab humanized IL-1β diabetes etc. GirentuximabRencarex mab chimeric carbonic anhydrase 9 clear cell renal cell (CA-IX)carcinoma[81] Glembatumumab vedotin mab human GPNMB melanoma, breastcancer Golimumab Simponi mab human TNF-α rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis Gomiliximab mab chimeric CD23 (IgEreceptor) allergic asthma Guselkumab mab human IL23 psoriasis Ibalizumabmab humanized CD4 HIV Ibritumomab tiuxetan Zevalin mab mouse CD20non-Hodgkin's lymphoma Icrucumab mab human VEGFR-1 cancer etc.Idarucizumab mab humanized dabigatran reversal of anticoagulant effectsof dabigatran Igovomab Indimacis-125 F(ab′)₂ mouse CA-125 ovarian cancer(diagnosis) IMAB362 mab human CLDN18.2 gastrointestinal adenocarcinomasand pancreatic tumor Imalumab mab human MIF cancer Imciromab Myoscintmab mouse cardiac myosin cardiac imaging Imgatuzumab mab humanized EGFRcancer Inclacumab mab human selectin P ? Indatuximab ravtansine mabchimeric SDC1 cancer Indusatumab vedotin mab human GUCY2C cancerInfliximab Remicade mab chimeric TNF-α rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, psoriasis, Crohn's disease, ulcerativecolitis Intetumumab mab human CD51 solid tumors (prostate cancer,melanoma) Inolimomab mab mouse CD25 (α chain of IL-2 graft versus hostdisease receptor) Inotuzumab ozogamicin mab humanized CD22 cancerIpilimumab Yervoy mab human CD152 melanoma Iratumumab mab human CD30(TNFRSF8) Hodgkin's lymphoma Isatuximab mab chimeric CD38 cancerItolizumab mab humanized CD6 ? Ixekizumab mab humanized IL-17Aautoimmune diseases Keliximab mab chimeric CD4 chronic asthmaLabetuzumab CEA-Cide mab humanized CEA colorectal cancer Lambrolizumabmab humanized PDCD1 antineoplastic agent Lampalizumab mab humanized CFD? Lebrikizumab mab humanized IL-13 asthma Lemalesomab mab mouse NCA-90(granulocyte diagnostic agent antigen) Lenzilumab mab human CSF2 ?Lerdelimumab mab human TGF beta 2 reduction of scarring after glaucomasurgery Lexatumumab mab human TRAIL-R2 cancer Libivirumab mab humanhepatitis B surface hepatitis B antigen Lifastuzumab vedotin mabhumanized phosphate-sodium co- cancer transporter Ligelizumab mabhumanized IGHE severe asthma and chronic spontaneous urticarialLilotomab satetraxetan mab mouse CD37 cancer Lintuzumab mab humanizedCD33 cancer Lirilumab mab human KIR2D ? Lodelcizumab mab humanized PCSK9hypercholesterolemia Lokivetmab mab veterinary Canis lupus familiaris ?IL31 Lorvotuzumab mab humanized CD56 cancer mertansine Lucatumumab mabhuman CD40 multiple myeloma, non- Hodgkin's lymphoma, Hodgkin's lymphomaLulizumab pegol mab humanized CD28 autoimmune diseases Lumiliximab mabchimeric CD23 (IgE receptor) chronic lymphocytic leukemia Lumretuzumabmab humanized ERBB3 cancer Mapatumumab mab human TRAIL-R1 cancerMargetuximab mab humanized ch4D5 cancer Maslimomab ? mouse T-cellreceptor ? Mavrilimumab mab human GMCSF receptor α- rheumatoid arthritischain Matuzumab mab humanized EGFR colorectal, lung and stomach cancerMepolizumab Bosatria mab humanized IL-5 asthma and white blood celldiseases Metelimumab mab human TGF beta 1 systemic sclerodermaMilatuzumab mab humanized CD74 multiple myeloma and other hematologicalmalignancies Minretumomab mab mouse TAG-72 tumor detection (andtherapy?) Mirvetuximab mab chimeric folate receptor alpha cancersoravtansine Mitumomab mab mouse GD3 ganglioside small cell lungcarcinoma Mogamulizumab mab humanized CCR4 cancer Morolimumab mab humanRhesus factor ? Motavizumab Numax mab humanized respiratory syncytialrespiratory syncytial virus virus (prevention) Moxetumomab mab mouseCD22 cancer pasudotox Muromonab-CD3 Orthoclone mab mouse CD3 preventionof organ OKT3 transplant rejections Nacolomab tafenatox Fab mouse C242antigen colorectal cancer Namilumab mab human CSF2 ? Naptumomabestafenatox Fab mouse 5T4 non-small cell lung carcinoma, renal cellcarcinoma Narnatumab mab human RON cancer Natalizumab Tysabri mabhumanized integrin α4 multiple sclerosis, Crohn's disease Nebacumab mabhuman endotoxin sepsis Necitumumab mab human EGFR non-small cell lungcarcinoma Nemolizumab mab humanized IL31RA ? Nerelimomab mab mouse TNF-α? Nesvacumab mab human angiopoietin 2 cancer Nimotuzumab Theracim, mabhumanized EGFR squamous cell Theraloc carcinoma, head and neck cancer,nasopharyngeal cancer, glioma Nivolumab mab human PD-1 cancerNofetumomab merpentan Verluma Fab mouse ? cancer (diagnosis)Obiltoxaximab mab chimeric Bacillus anthracis Bacillus anthracis sporesanthrax Obinutuzumab Gazyva mab humanized CD20 Chronic lymphaticleukemia Ocaratuzumab mab humanized CD20 cancer Ocrelizumab mabhumanized CD20 rheumatoid arthritis, lupus erythematosus etc. Odulimomabmab mouse LFA-1 (CD11a) prevention of organ transplant rejections,immunological diseases Ofatumumab Arzerra mab human CD20 chroniclymphocytic leukemia etc. Olaratumab mab human PDGF-R α cancerOlokizumab mab humanized IL6 ? Omalizumab Xolair mab humanized IgE Fcregion allergic asthma Onartuzumab mab humanized human scatter factorcancer receptor kinase Ontuxizumab mab chimeric/humanized TEM1 cancerOpicinumab^(]) mab human LINGO-1 multiple sclerosis Oportuzumab monatoxscFv humanized EpCAM cancer Oregovomab OvaRex mab mouse CA-125 ovariancancer Orticumab mab human oxLDL ? Otelixizumab mab chimeric/humanizedCD3 diabetes mellitus type 1 Otlertuzumab mab humanized CD37 cancerOxelumab mab human OX-40 asthma Ozanezumab mab humanized NOGO-A ALS andmultiple sclerosis Ozoralizumab mab humanized TNF-α inflammationPagibaximab mab chimeric lipoteichoic acid sepsis (Staphylococcus)Palivizumab Synagis, mab humanized F protein of respiratory syncytialAbbosynagis respiratory syncytial virus (prevention) virus PanitumumabVectibix mab human EGFR colorectal cancer Pankomab mab humanized tumorspecific ovarian cancer glycosylation of MUC1 Panobacumab mab humanPseudomonas Pseudomonas aeruginosa aeruginosa infection Parsatuzumab mabhuman EGFL7 cancer Pascolizumab mab humanized IL-4 asthma Pasotuxizumabmab chimeric/humanized folate hydrolase cancer Pateclizumab mabhumanized LTA TNF Patritumab mab human HER3 cancer Pembrolizumab mabhumanized PDCD1 cancer etc. Pemtumomab Theragyn ? mouse MUC1 cancerPerakizumab mab humanized IL17A arthritis Pertuzumab Omnitarg mabhumanized HER2/neu cancer Pexelizumab scFv humanized C5 reduction ofside effects of cardiac surgery Pidilizumab mab humanized PD-1 cancerand infectious diseases Pinatuzumab vedotin mab humanized CD22 cancerPintumomab mab mouse adenocarcinoma adenocarcinoma antigen (imaging)Placulumab mab human human TNF ? Polatuzumab vedotin mab humanized CD79Bcancer Ponezumab mab humanized human beta-amyloid Alzheimer's diseasePriliximab mab chimeric CD4 Crohn's disease, multiple sclerosisPritoxaximab mab chimeric E. coli shiga toxin ? type-1 Pritumumab mabhuman vimentin brain cancer PRO 140 ? humanized CCR5 HIV infectionQuilizumab mab humanized IGHE asthma Racotumomab mab mouseN-glycolylneuraminic cancer acid Radretumab mab human fibronectin extracancer domain-B Rafivirumab mab human rabies virus rabies (prophylaxis)glycoprotein Ralpancizumab mab humanized neural apoptosis- dyslipidemiaregulated proteinase 1 Ramucirumab Cyramza mab human VEGFR2 solid tumorsRanibizumab Lucentis Fab humanized VEGF-A macular degeneration (wetform) Raxibacumab mab human anthrax toxin, anthrax (prophylaxis andprotective antigen treatment) Refanezumab mab humanizedmyelin-associated recovery of motor glycoprotein function after strokeRegavirumab mab human cytomegalovirus cytomegalovirus glycoprotein Binfection Reslizumab mab humanized IL-5 inflammations of the airways,skin and gastrointestinal tract Rilotumumab mab human HGF solid tumorsRinucumab mab human platelet-derived neovascular age-related growthfactor receptor macular degeneration beta Rituximab MabThera, mabchimeric CD20 lymphomas, leukemias, Rituxan some autoimmune disordersRobatumumab mab human IGF-1 receptor cancer Roledumab mab human RHD ?Romosozumab mab humanized sclerostin osteoporosis Rontalizumab mabhumanized IFN-α systemic lupus erythematosus Rovelizumab LeukArrest mabhumanized CD11, CD18 haemorrhagic shock etc. Ruplizumab Antova mabhumanized CD154 (CD40L) rheumatic diseases Sacituzumab govitecan mabhumanized tumor-associated cancer calcium signal transducer 2Samalizumab mab humanized CD200 cancer Sarilumab mab human IL6rheumatoid arthritis, ankylosing spondylitis Satumomab pendetide mabmouse TAG-72 cancer (diagnosis) Secukinumab mab human IL-17A uveitis,rheumatoid arthritis psoriasis Seribantumab mab human ERBB3 cancerSetoxaximab mab chimeric E. coli shiga toxin ? type-2 Sevirumab ? humancytomegalovirus cytomegalovirus infection Sibrotuzumab mab humanized FAPcancer SGN-CD19A mab humanized CD19 acute lymphoblastic leukemia andB-cell non- Hodgkin lymphoma SGN-CD33A mab humanized CD33 Acute myeloidleukemia Sifalimumab mab humanized IFN-α SLE, dermatomyositis,polymyositis Siltuximab mab chimeric IL-6 cancer Simtuzumab mabhumanized LOXL2 fibrosis Siplizumab mab humanized CD2 psoriasis,graft-versus- host disease (prevention) Sirukumab mab human IL-6rheumatoid arthritis Sofituzumab vedotin mab humanized CA 125 ovariancancer Solanezumab mab humanized beta amyloid Alzheimer's diseaseSolitomab mab mouse EpCAM ? Sonepcizumab ? humanized sphingosine-1-choroidal and retinal phosphate neovascularization Sontuzumab mabhumanized episialin ? Stamulumab mab human myostatin muscular dystrophySulesomab LeukoScan Fab mouse NCA-90 (granulocyte osteomyelitis(imaging) antigen) Suvizumab mab humanized HIV-1 viral infectionsTabalumab mab human BAFF B-cell cancers Tacatuzumab tetraxetan AFP-Cidemab humanized alpha-fetoprotein cancer Tadocizumab Fab humanizedintegrin alIbra percutaneous coronary intervention Talizumab mabhumanized IgE allergic reaction Tanezumab mab humanized NGF painTaplitumomab paptox mab mouse CD19 cancer[citation needed] Tarextumabmab human Notch receptor cancer Tefibazumab Aurexis mab humanizedclumping factor A Staphylococcus aureus infection Telimomab aritox Fabmouse ? ? Tenatumomab mab mouse tenascin C cancer Teneliximab mabchimeric CD40 ? Teplizumab mab humanized CD3 diabetes mellitus type 1Teprotumumab mab human CD221 hematologic tumors Tesidolumab mab human C5? TGN1412 ? humanized CD28 chronic lymphocytic leukemia, rheumatoidarthritis Ticilimumab (= mab human CTLA-4 cancer tremelimumab)Tildrakizumab mab humanized IL23 immunologically mediated inflammatorydisorders Tigatuzumab mab humanized TRAIL-R2 cancer TNX-650 ? humanizedIL-13 Hodgkin's lymphoma Tocilizumab[6](= Actemra, mab humanized IL-6receptor rheumatoid arthritis atlizumab) RoActemra Toralizumab mabhumanized CD154 (CD4OL) rheumatoid arthritis, lupus nephritis etc.Tosatoxumab mab human Staphylococcus aureus ? Tositumomab Bexxar ? mouseCD20 follicular lymphoma Tovetumab mab human CD140a cancer Tralokinumabmab human IL-13 asthma etc. Trastuzumab Herceptin mab humanized HER2/neubreast cancer TRBS07 Ektomab 3funct ? GD2 melanoma Tregalizumab mabhumanized CD4 ? Tremelimumab mab human CTLA-4 cancer Trevogrumab mabhuman growth differentiation muscle atrophy due to factor 8 orthopedicdisuse and sarcopenia Tucotuzumab mab humanized EpCAM cancer celmoleukinTuvirumab ? human hepatitis B virus chronic hepatitis B Ublituximab mabchimeric MS4A1 cancer Ulocuplumab mab human C-X-C chemokine hematologicreceptor type 4 malignancies Urelumab mab human 4-1BB cancer etc.Urtoxazumab mab humanized Escherichia coli diarrhoea caused by E. coliUstekinumab Stelara mab human IL-12, IL-23 multiple sclerosis,psoriasis, psoriatic arthritis Vandortuzumab vedotin mab humanizedSTEAP1 cancer Vantictumab mab human Frizzled receptor cancer Vanucizumabmab humanized angiopoietin 2 cancer Vapaliximab mab chimeric AOC3(VAP-1) ? Varlilumab mab human CD27 ? Vatelizumab mab humanized ITGA2 ?Vedolizumab mab humanized integrin α4β7 Crohn's disease, ulcerativecolitis Veltuzumab mab humanized CD20 non-Hodgkin's lymphoma Vepalimomabmab mouse AOC3 (VAP-1) inflammation Vesencumab mab human NRP1 ?Visilizumab Nuvion mab humanized CD3 Crohn's disease, ulcerative colitisVolociximab mab chimeric integrin α5β1 solid tumors Vorsetuzumabmafodotin mab humanized CD70 cancer Votumumab HumaSPECT mab human tumorantigen colorectal tumors CTAA16.88 Zalutumumab HuMax-EGFr mab humanEGFR squamous cell carcinoma of the head and neck Zanolimumab HuMax-CD4mab human CD4 rheumatoid arthritis, psoriasis, T-cell lymphoma Zatuximabmab chimeric HER1 cancer Ziralimumab mab human CD147 (basigin) ?Zolimomab aritox mab mouse CD5 systemic lupus erythematosus, graft-versus-host disease

In another embodiment using photoacoustic/thermoacoustic technology, thecirculating tumor, exosomes, or extracellular vesicles in the blood arequantified non-invasively by having a thermal energy source such aslaser microwave, RF, or other unit mounted on the patient's wrist, neck,etc. and a receiver to count and record the sound wave generated bycirculating cells to which the antibody-coated nanoparticles areattached.

In another embodiment, the ultrasonic receiver of the photoacoustic unitis an array of ultrasonic receivers mounted on a hand held probe. Thehand held probe contacts the patient's skin via a gel placed over thearea suspected to contain a tumor or lesion. It simultaneously recordsmultiple photoacoustic signals from the lesion during thermal energyapplication. Thermal energy applied pulses can range from one per secondto a million times or more per second. Each time a thermal pulse reachesthe nanoparticles, the nanoparticles expand and create aphotoacoustic/thermoacoustic response that is recorded by thephotoacoustic receiver.

The probe can be moved in any direction, e.g., up and down, side toside, etc., over the skin while recording the sound waves from thenanoparticles. Using a processor in the photoacoustic/thermoacousticunit, one uses the photoacoustic response data to construct a two- orthree-dimensional image of the tumor. The hand held probe permitsscanning any bodily surface, including but not limited to breast, eye,CNS, spinal cord, extremities, internal organs, eye, nose, chest,trachea, throat, abdomen, and urogenital organs. The data from theultrasonic array probe of the photoacoustic/thermoacoustic unit isstored in a computer during the probe's motion, permitting videoconstruction showing tumor shape, structure, location, etc. for videopresentation, evaluation, and archiving.

In one embodiment, the unit is capable of storing vast quantities ofdata from photoacoustic signals. The unit is also capable of storingvast quantities of data from non-stationary tissues, e.g., circulatingtumor cells and exosomes in blood vessels, that have accumulatedantibody coated nanoparticles on their cell membranes. The targetedcells can also be any normal or abnormal circulating cell in the bloodor lymphatic system. The photoacoustic unit reproduces signals fromthese mobile cells and/or exosomes as photoacousticcinematography/angiography or video.

In one embodiment, the cinematography or video recording is done by thephotoacoustic unit recording at least 30 frames/second of photoacousticsignals, and converting them into an image of a moving object. Acinematography or video is performed by obtaining at least 30 frames ofphotos of a moving object per second. In photoacoustic videography orphotoacoustic angiography, 30 or more frames of pulse signals from theheated nanoparticles per second are needed to reproduce or convert thestill images to a moving object, e.g., blood flow, etc. by the unit. Useof such a system is known: Peyman et al. Ophthalmic Surg Laser Imaging43 (2012) 143-51 doi: 10.3928.15428877-20120105-01 showing, however,lower resolution because no nanoparticles or photoacoustic imagingsystem was employed, and expressly incorporated by reference herein inits entirety.

In one embodiment the photoacoustic processor converts the microscopicstill images to a video or photoacoustic angiography; since the onlymoving parts in the vessels that are targeted with antibody coatednanoparticles are the circulating tumor cells or exosomes, extracellularvesicles or bubbles covered with antibody coated nanoparticles that areheated by a pulse of thermal energy produces an internal ultrasonicpulse signal recorded by the photoacoustic receiver. A moving image ofthe cells and exosomes can be created by the unit whether the cells areon the tumor interior or exterior.

Nanoparticle assisted photoacoustic video-angiography or nanoparticleassisted photoacoustic cinematography is novel and inventive. All“photoacoustic” terminology has previously been used for describingtissue heating or the difference in the temperature between two tissues,vessels vs. skin, and has been done with light alone, not in combinationwith nanoparticles. In one embodiment, the method is performed fortherapy by providing the patient with at least one antibody-coatedfunctionalized nanoparticle having a detectable property, with theantibody targeting the functionalized nanoparticle to a specific patientsite, then heating the nanoparticles to generate a photoacoustic signal,i.e., thermal therapy, and imaging to visualize any localizednanoparticle at the site. The ultrasonic receiver of the photoacousticunit is an array of ultrasonic receivers mounted on a hand held probesimultaneously recording multiple photoacoustic signals from the lesionduring thermal energy application which in one embodiment is pulsating.The array of ultrasonic receivers of the photoacoustic unit mounted on ahand held probe simultaneously records multiple photoacoustic signalsfrom the lesion or vessels during thermal energy application,reproducing motion of moving nanoparticles and/or cells as ananoparticle assisted photoacoustic video-angiography or nanoparticleassisted photoacoustic cinematography.

In another embodiment, software associated with thephotoacoustic/thermoacoustic unit can enhance either or both thephotoacoustic signals and resulting images. Enhancement may facilitatedifferentiating exosomes from circulating cells due to the smallerexosome size. All exosomes or other types of extracellular vesicles areless than one micron; in contrast, tumor cells are five to twenty timeslarger than exosomes. The inventive system for the first time permits invivo observation and separation of exosomes from tumor cells, andseparation of circulating tumor cells from a tumor mass. The separatedcells or cell structures can be observed, counted, and quantified toassess the therapeutic effect of a procedure on tumor cells.

In another embodiment, after imaging and therapy, the biomarkers arecollected from liquid biopsies and compared with those obtained prior totherapy in different post-operative periods to confirm the therapeuticeffect of the procedure and prognosticate the condition.

In another embodiment, the antibody coated nanoparticles are conjugatedand administered with checkpoint inhibitors along with known immunetherapy agents and vaccines to facilitate circulating killer cellsattack and removal of tumor cells.

In another embodiment, the vaccines with or without VLP facilitatecirculating killer cells attacking and removal of tumor cells, and theantibody coated nanoparticles are administered with checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.and Rock inhibitors (e.g., Fasudil or Botox) or Wnt inhibitor, such asniclosamide, ivernectin, etc., and for the future management of thetumor recurrences in the patient or treatment of metastatic disease.

In another embodiment, polymeric nanoparticles or polysaccharide orsynthetic polymers conjugated with biomarkers are administered toenhance a vaccination effect and are taken up by antigen presentingcells.

In one embodiment, genetic analysis of the patient is performed todetermine a sequence of the gene that is mutated. A sample of thepatient's blood is analyzed for any of the following indicia of thepresence or a neoplasm or a predisposition to a neoplasm: specific tumorbiomarker(s), non-specific tumor biomarker(s), extravascular vesicles,circulating tumor cells, tumor micro RNA, micro DNA, or any other tumorindicator. RNA sequencing reflects the dynamic nature of gene expressionfor detection of RNA fragments, including mRNA, noncoding RNA, chimericRNA, pathogen RNA, extracellular RNA, etc.

Examples of biomarkers have been previously disclosed. Other biomarkersinclude DNA hypermethylation, the presence of ZNF154 in colon, lung,breast, stomach, and endometrial tumor, and the stem cell marker NANOG,a mitochondrial oxidative phosphorylation/fatty acid oxidation moleculein highly malignant tumor-initiating stem-like cells (TICs) thatreprograms mitochondrial metabolism.

Use of results from a patient's genetic analysis advantageously permitsselection of a therapeutic agent, along with antibody-coatednanoparticles conjugated with thermosensitive polymers andthermotherapy, to provide the greatest efficacy against cancers that aresmaller than 4 mm in diameter. In general, such cancers have not grownto a size whereby they show genetic differentiation of the cancer cells.Treatment of these small cancer cells can thus include treatment of thecancer stem cell(s). In one embodiment, nanoparticles activated byelectromagnetic radiation, either in vitro or in vivo, enhance both genetransfer and cell proliferation of any desired cell, including stemcells

In one embodiment, the patient's blood is processed to isolate thepatient's own natural killer (NK) cells, i.e., a type of lymphocyte thatis part of the patient's innate immune system, and dendritic cells,i.e., immune cells that process antigen material and present it on thecell surface to T cells of the immune system). NK cells and dendriticcells are isolated from a patient's blood using commercially availablekits known in the art, e.g., EasySep™ and RosetteSep™ STEMCELLTechnologies Inc., Tukwila Wash.; NK Cell Isolation Kit, MeltinyiBiotech, Bergisch Gladbach Germany. The natural killer cells/dendriticcells are rendered sensitized to the tumor in vitro. Sensitization isaccomplished by co-culturing the patient's natural killer cells and/ordendritic cells with IL-2 and the antibody-coated nanoparticlescontaining the optional penetration-enhancing agents and/orthermosensitive polymers as previously described. The patient'ssensitized natural killer cells/dendritic cells are then administered tothe patient at intervals to provide a booster immune therapy, much as avaccine booster injection does. The vaccine may be administered with orwithout VLPs. IL-2 is a protein produced by the T cells. When IL-2 isconjugated with the thermosensitive antibody coated nanoparticle, andadministered with checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor, 15D11, etc. and Rock inhibitors, such as Fasudil orBotox, or Wnt inhibitors, such as niclosamide and ivermectin, or smallmolecule Wnt inhibitors upon controllable temperature release, IL-2 issystemically available to enhance a T-cell response in the patient bycell sensitization and proliferation as a vaccine or treatment ofmetastatic disease.

Thermal damage to the tumor cell membrane as a part of nanoparticleassisted thermotherapy releases antigens that, in vivo, activate andstimulate a dendritic cell immunogenic response. The activated dendriticcells induce a signal that additionally activates T cell-driven tumorcell damage or killing.

In one embodiment, the medium used to culture NK/dendritic cellscontains viral like particles (VLP). The NK/dendritic cells pick up theVLP and enhance sensitization against the tumor. If tumor cell biopsyspecimens are available, NK cells/dendritic cells are cultured fromthese biopsy specimens which additionally contain tumor lysate, killedcirculating tumor (CT) cells, and their extracellular vesicles (ECV). Inone embodiment, nanoparticles with thermosensitive polymers andconjugated with tumor antibody and VLP are administered to the patientintravenously, as the first step of tumor vaccination and therapy. Thenanoparticles become attached to the tumor cells within a few minutesafter administration.

In one embodiment, the tumor biomarkers from a patient's blood areidentified, and anti-tumor antibodies are prepared, using conventionalantibody techniques known in the art. The antibodies may be monoclonal,polyclonal, humanized, etc.; tumor antibodies also includes aptamers(oligonucleotide or peptides that bind to a specific target). Theantibodies/aptamers are then coated on diagnostic or therapeuticnanoparticles or quantum dots, and conjugated with checkpoint inhibitorssuch as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rockinhibitors, such as Fasudil, hydroxyl Fasudil, etc. botox, 3C exoenzymeetc. or Wnt inhibitor, such as niclosamide, as a vaccine or treatment ofmetastatic disease. which are then systemically administered to thepatient. In vivo, the tumor-antibody-coated nanoparticles seek the tumorcells via the specificity of the anti-tumor antibody component. In oneembodiment, adding a cell penetration enhancing agent to the polymer orother coating facilitates penetration of the tumor-antibody-coatednanoparticles into a tumor cell. Cell-penetration enhancing agentsrender the nanoparticle complex more biocompatible, and have beenpreviously disclosed; they include cell penetrating peptide (CPP),activated CPP (APCC), (poly)ethylene glycol (PEG), biotin streptavidin,etc.

In one embodiment, as previously disclosed, the tumor-antibody-coatednanoparticles are also coated with a thermosensitive polymer thatdissolves at a particular temperature, e.g., a polymer such as chitosanthat dissolves at a temperature of 40° C.-43° C., and/or an argininerich polymer, etc.

This coating, in addition to its thermosensitive properties, isadministered with checkpoint inhibitors such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, such as Fasudil,Botox, 3C exoemzyme, etc. or Wnt inhibitor, such as niclosamide, etc.,and includes one or more medicaments, genes, etc. thus providingadditional therapy to the patient upon administration and thermotherapyas a vaccine or treatment of metastatic disease. In one embodiment,adding a phospholipase, anti-phospholipid antibody, toxin (snake,scorpion, bee venom, etc. to the polymer or other coating enhances thedamage to the cell membrane from an anti-tumor antibody coatednanoparticle. This beneficially increases the hyperthermal damage tocancer and other undesirable cells due to toxin release from thenanoparticles' coating of thermosensitive polymer at 40° C.-43° C.

In one embodiment, genes are provided that have a stimulatory action inresponse to light or ultrasound. An example of such a gene is the opsingene and members of the opsin family. In this embodiment, such genes areprovided to regulate cell membrane polarization and depolarization. Suchgenes can thus controllably create an action potential in the membraneof an excitable cell, such as a retinal cell, or a non-excitable cellsuch as a tumor cell. Controllable regulation may drive a permanentdepolarization state to render the cells accessible to a desiredmedicament for cell destruction.

In one embodiment, combinations of genes can be used for controllableregulation. As an example, genes responding to light to produce actionpotential, combined with genes that can modifying a defective gene(s) inthe cells of an organ, e.g., eye, brain, lung, spinal cord, peripheralnerve, lung, digestive tract, can be used in combination to facilitateregulation of actions including swallowing, breathing, gland secretion,etc., to restore the normal function of the organ. As another example,genes responding to light to produce an action potential, combined withinhibitory genes such as siRNA, RNAi, microRNA, can be used to inhibittumor function by simultaneous depolarization of the tumor cells. Thesegenes can additionally be combined with chemotherapeutic agent to worksynergistically and damage the tumor cells.

Systemic administration of tumor antibody coated nanoparticles, coatedwith thermosensitive polymers and a cell penetration facilitating agent,targets the nanoparticles toward the tumor cell membrane. Externalenergy is applied by a thermal delivery device that uses energy(electromagnetic radiation, microwave radiation, radiofrequency waves,an alternating magnet, focused ultrasound, etc.) to increase thetemperature of the nanoparticle. The heated nanoparticle absorbs moreenergy than the tissue surrounding the nanoparticle. The temperatureincrease causes the nanoparticles to expand. Expansion of thenanoparticles creates a photoacoustic, thermoacoustic, or ultrasoundwave, whose sound wave amplitude correlates with the amount of thetemperature increase, i.e., the degree of the temperature rise.

In one embodiment, the ultrasound wave is recorded by a transducer andis transmitted to a unit to image the nanoparticle increase intemperature as one-, two- or three-dimensional images. This unit isconnected to the thermal delivery device via a computer to maintain theamount of thermal energy needed for the time required to heat thenanoparticles to the desired temperature and for the desired time periodand thus release medicament(s), gene(s), VLP, etc. These agents may alsobe against microorganisms, e.g., bacterial, viral, fungal, or parasiticagents, that have developed resistance to the therapeutic agents. Forexample, heated bacteria become more permeable to diffusion ofappropriate medication; in contrast, non-heated bacteria remainresistant.

In one embodiment, nanoparticles coated with the desired antibody (e.g.,anti-tumor antibody, anti-bacterial protein antibody, etc.) areadministered to the patient to assure that the antibody-nanoparticlecomplex is in contact with the appropriate cells or tissues. It will beappreciated that the appropriate cells or tissues may include bothcirculating cells (e.g., ECV, endosomes, leukemic cells, etc.) andnon-circulating cells (e.g., solid tumor).

In one embodiment, a small hand held photoacoustic unit with a smallthermal delivery unit e.g. laser, microwave, or radiofrequency unit isplaced externally over a subcutaneously located vessel to deliver apulse of energy and to heat the nanoparticles attached to thecirculating tumor cells and create a photoacoustic sound as they heatup. This records the sound wave each time a tumor cell passes by theexternal hand held unit, adjusts the temperature from 37° C.-43° C.,thus assessing and quantifying non-invasively the circulating tumorcells using the hand held thermal imaging device.

In one embodiment, photoacoustic technology imaging is controlled to alow temperature of 37° C.-43° C., thus assessing and non-invasivelymeasuring circulating cells using a hand held thermal imaging device.Imaging may be used in combination with any standard method, includingbut not limited to radiography, computed tomography (CT), magneticresonance imaging (MRI), ultrasound, positron and other molecularimaging devices.

In one embodiment, nanoparticles are conjugated with VLP derived fromplant viruses. In this embodiment, the VLP are used for cancer therapyby carrying sRNA, RNAi, etc. The host of these viruses are plants e.g.tobacco mosaic virus (TMV), bean yellow dwarf virus (BeYDV), etc., whichcannot infect the patient. Thermal application of the antibody coatednanoparticles provides the control over when and where these particlesare released to provide maximum benefit in immunotherapy. The VLP aregenerally immunogenic and do not require adjuncts to induce an immuneresponse. These modified viruses are devoid of genetic components andcannot replicate in the body. However, if a specific gene of a specificprotein e.g. an antibody, is conjugated with them and injected in theplant, the modified viruses produce large amounts of the antibody orprotein in the plant, which can subsequently be extracted and used inhuman infective or non-infective diseases or to produce a vaccine totreat e.g. Alzheimer's disease etc. The thermal application at 41-43degrees C. damages the VLP or other viruses and prevents theirproliferation without reducing their immunogenicity. Once the antibodyis produced, it can be used in diagnosing or guiding treatment to theaffected area in combination with nanoparticles with or without VLP anddrug delivery, and administering them with Rock inhibitors, such asFasudil, botox 3 C exoenzyme, etc., or Wnt inhibitors, such asniclosamide to be used as a vaccine or treatment of metastatic diseasesneeded.

With respect to a gene(s) present in the polymer coating, e.g., aninhibitory gene such as siRNA, siDNA, RNAi, or an appropriate checkpointinhibitor, may be used. Checkpoint inhibitors enhance cellular immuneresponses to tumor specific proteins in the cancer cells, as previouslydisclosed. In one embodiment, checkpoint inhibitors, such as PD-1,PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, suchas Fasudil, botox or Wnt inhibitors, such as niclosamide or ivermectin,as a vaccine or for treatment of metastatic diseases needed or arecombined with nanoparticle-assisted targeted immunotherapy as anadaptive T-cell transfer mechanism. These, along with the a CRISPR/cas9or CRISPR interference (CRISPRi) complex, may perturb or modify thetumor genes.

With respect to a medicament(s), the medicament(s) would be releasedlocally present in the polymer coating or pluralities of the antibodycoated nanoparticles during thermotherapy. For a medicament that is abiologic, local release permits agents to be concentrated at the desiredsite without being released systemically resulting in systemic toxicitythe medicament may otherwise cause. As one example, anti VEGF agents,TNF inhibitors, antineoplastic medications such as taxol,antimetabolites, anti-inflammatory agents, steroids, checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.and Rock inhibitors, such as Fasudil, 3C exoenzyme, Botox, etc., or Wntinhibitors, such as niclosamide or ivermectin, antibiotics, antiviralagent, etc. can be used as a vaccine or for treatment of metastaticdiseases and become localize specifically at a tumor or other site atsignificantly higher concentrations to stop tumor neovascular growth ordamage the tumor etc., without causing the known systemic complicationssuch as heart attack, intestinal bleeding, kidney disease, liverdisease, or suppressing the normal humoral or cellular immune responseof the body, etc. as seen in routine chemotherapy or immune therapy. Asanother example, release of phospholipase enzymes can create a hole inthe membrane of tumors or other cells to provide or facilitate entry ofa medicament(s) and/or gene(s) entry into a cell.

In the inventive precision nanoparticle assisted thermotherapy imaging(NATTI), the temperature of the tumor cell to which the nanoparticle isconjugated is controllably precisely increased. The temperatureincreases (a) releases a medicament(s) and/or gene(s) from athermosensitive coating on the nanoparticle, and (b) enhancespenetration of the medicament(s) and/or gene(s) through the open poresof the tumor cell membrane. NATTI technology includes acomputer-controlled thermal energy delivery unit to ensure attainment ofa desired increased temperature of the tumor for achieving thetherapeutic goal. Controlling thermal energy delivery to achieve atemperature from 38° C. to 42° C. for drug delivery or more in thetumor-nanoparticle complex to a tumor, or to another tissue affected bya disease as directed by antibody binding to a corresponding antigen. Itwill be appreciated that the increased temperature may be maintained atthe controlled desired level for any desired time interval, e.g., up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes, or evenlonger, depending upon the need.

Typically, normal healthy cell membranes are comprised of thephospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE),which are located within the inner membrane and oriented toward the cellinterior. However, for cancer cells, the orientations of PS and PE areflipped, so each oriented toward the cell exterior. The melting point ofPS and PE is about 65° C.-70° C. degree. When the nanoparticles areheated to this temperature, the exposed PS and PE lipids of the cellmembrane melt, and create a dehiscence in the cell membrane throughwhich chemotherapeutic agents can freely flow into the tumor cell,killing the tumor cell.

For a medicament that is a chemotherapeutic agent, local release permitsa higher concentration of a chemotherapeutic agent to be contained inthe polymer coating, i.e., a supratherapeutic concentration, because itis confined to a localized site and would not result in systemictoxicity, yet still would achieve a higher therapeutic level at thetumor site. In one embodiment, a known existing antitumorchemotherapeutic agent is administered at a concentration that exceedsthat of a concentration that would be administered under typicaltherapy, yet that does not result in patient toxicity. Similarly, onecan use a toxic medicament(s) to locally perturb a cellular metabolicpathway or a specific cell cycle, e.g., local tumor cell perturbation.Such agents are generally not administered intravenously or orallybecause of their serious or fatal systemic toxicity and side effects.The concentration of the chemotherapeutic medicament(s) delivered by theinventive method is, in general, high locally, but less the 1/1000 ofthe concentration that would be required if the medicament(s) were to beeffective if delivered systemically.

Use of precision nanoparticle assisted thermotherapy and imaging (NATTI)may be used to fine-tune the approach to perturb survival mechanisms oftumors or other pathological cells. It will be thus appreciated that theinventive method may be used as a rapid en mass treatment of a cancer oran organ. For example, it may be used as a preliminary treatment inadvance of other therapies that in general have severe debilitatingsystemic complications such as immune suppression, etc. and which maytake longer to obtain approval for their administration. Thus,nanoparticle assisted thermotherapy and drug delivery NATTI avoids thechemotherapy complication of damaging the patient's immune system as aresult of one or multiple chemotherapeutic agents used in these latemetastatic cancer patients.

The nanoparticle assisted thermoacoustic imaging technology, along withthermal energy, drives the medicament(s), gene(s), VLP into the cancercell, with simultaneous generation of an immune response to the cancercell and inhibition of cancer cell proliferation by including siRNA,siDNA, etc., along with simultaneously enhancing gene therapy byconjugating a CRISPR/cas9 or CRISPRi complex to the nanoparticle tocorrect or inhibit a genetic component of a tumor. Once inside a cell,e.g., a tumor cell or the cell of an organ, the gene(s), along with theCRISPR/cas9 complex or CRISPRi, enter the cell nucleus or mitochondriaand precisely modify the gene pool of those cells. The RNA-guidedbacterial endonuclease Cas9 is the effector protein of the type IICRISPR/Cas9 system that detects and subsequently generates adouble-strand break (DSB) in target DNA. This may treat a disease causedby a gene deficiency, or add a new useful gene(s), or remove andpossible replace a gene, in the cell nucleus or the mitochondria.

The gene(s) and/or medicaments(s) may be delivered to a specific site,but not released in the circulation from the nanoparticles untilreaching the required elevated temperature and after attaching to thetumor or other desired cells or desired location. It will be appreciatedthat the inventive method can be used in therapy for non-neoplasticdiseases. As one example, the amyloid plaques present in Alzheimer'sdisease may be used to produce anti-amyloid plaque antibodies andtreated by the inventive method. As another example, bacteria inpatients with severe sepsis refractory to standard antibiotic therapy,e.g., patients with methicillin-resistant Staphylococcus aureus (MRSA)etc., may be used to produce anti-bacterial antibodies and treated bythe inventive method. In this example, the method may be combined withextracorporeal treatment of blood, using a thermal energy delivery unitto provide electromagnetic radiation, radiofrequency waves, microwaves,focused ultrasound, an alternating magnetic field, etc., under thecontrol of the described NATTI unit, to controllably achieve atemperature of 42° C.-45° C. to kill the bacteria prior to cooling theblood to the normal 37° C. prior to reinfusion to the patient.

In addition, increasing the temperature of the nanoparticlesincrementally from 37° C.-43° C. allows precision nanoparticle assistedthermotherapy and imaging (NATTI) to release a gene(s) or medicament(s)from the nanoparticles. It will be appreciated that the methodbeneficially permits imaging a tumor or other desired cells, such asAlzheimer's plaques, that are present in a small lesion otherwiseinvisible by conventional imaging methods such as radiography.

The immune response is generated by two different mechanisms. Onemechanism is by releasing the VLP, which then stimulate a cellularimmune response at the site of the tumor. The other mechanism is by thethermotherapy-damaged tumor cells releasing their antigenic material inand beyond the surrounding tissues, creating a more active cellularimmune response due to the additional tumor antigens present, anddrawing the patient's immune cells, dendritic cell, T-lymphocytes,B-lymphocytes, macrophages, etc., to the tumor location. This mechanismalso advantageously provides immune memory functioning as an internalvaccination method. Specifically, local release of antigens from damagedtumor cells enhances a patient's immune response to a large amount ofother tumor cell associated antigens, creating a form of in vivovaccination. Such vaccination can be provided as needed, e.g., annually,at specific intervals, upon specific events, etc. to prime the patient'simmune cells against any future tumor cells, and protects againstreappearance of any tumors with similar antigenic components. Forexample, vaccination may be administered annually or biannually orbetween annual and biannual administration indefinitely, unless newbiomarkers are discovered in the patient, necessitating additionaland/or earlier therapy.

In addition, the inflammatory process created as a result of the immunetherapy and cellular response increases the temperature of the tissueinvolved, which is also recorded using photoacoustic technology imagingto image the tumor location and its potential metastatic lesionsanywhere in the body.

This embodiment results in precise, local, internally-inducedimmunotherapy and simultaneous vaccination. The antigen, e.g. VLP, isdelivered intravenously with thermosensitive polymers conjugated withantitumor antibody coated nanoparticles. The VLP are released from thesenanoparticles only when the temperature of the nanoparticle isincreased, and the nanoparticles are localized only at the tumor site,due to the specificity of the anti-tumor antibody with which thenanoparticles are conjugated.

As previously disclosed, various nanoparticle types, compositions,configurations, etc. are possible, including the following non-limitingexamples: organic, synthetic, metallic, non-metallic, magnetic,non-magnetic, paramagnetic, etc., configurations such as a nanosphere,nanotube, nanoshell, nanocage, nanocarbon, etc., including quantum dots,dendrimers, liposomes, piezoelectric nanoparticles, etc.

In one embodiment, piezoelectric nanoparticles are stimulated by anultrasonic unit, providing a therapeutic effect by inducing an electriccurrent in cells. Depending upon the frequency, this exposure can killcells on one hand, or it can enhance growth of specific cells on theother hand. Application of thermal energy at a frequency in the range of1 Hz-20 Hz promotes cell growth. Application of thermal energy at afrequency greater than about 60 Hz damages cells. Cell death isdesirable for treating pathologies such as cancer. However, cellproliferation is desirable to facilitating tissue regeneration. Forexample, in this embodiment, a patient with a stroke, or a myocardialinfarction, or a spinal cord injury, may be treated to regenerate brain,heart, nerve tissue respectively. In this embodiment, the antibody usedis targeted to the damaged cells, i.e., neurons, cardiac cells, etc.,and treatment is with a pulsed frequency of 1 Hz-20 Hz or more isprovided for 1 min-10 min. It will be appreciated that this embodimentpermits stem cells to be controllably either stimulated or inhibited.

In one embodiment, the nanoparticle stimulates proliferation of in vitrocultured cells when the nanoparticle is exposed to and absorbs lightpulses of low frequency, i.e., frequencies in the range of 1 Hz-20 Hz.Conversely, in one embodiment, the nanoparticle inhibits cellproliferation when the nanoparticle is exposed to and absorbs lightpulses of very high frequency, i.e., frequencies in the range of >30Hz-100 Hz). Thus, selecting the frequency of the thermotherapy, and thusthe frequency to which the tumor antibody-coated nanoparticles areexposed, adds to the mechanisms of therapy the patient receives if thelight pulses are at low frequencies, i.e., no higher than about 20 Hz.

In one embodiment, after sensitization of the immune cells with thetumor antigen, functionalized quantum dots with antibody coated againstcell membrane of immune cells is added so that the cell membranes of theimmune cells carry a marker that can be made visible with specificwavelength of light extracorporally.

In one embodiment, antibody coated nanoparticles are conjugated withthermosensitive polymers containing VLP/medication/genes, andadministered with checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, such as Fasudil,botox, or Wnt inhibitors, such as niclosamide etc., and intravenouslyadministered to a patient at far lower level than would be toxic to thebody (i.e., 1/10 to 1/100 of the non-toxic dose approved by the FDA).The VLP are released from the thermosensitive nanoparticles by thermalapplication at temperatures of 41° C.-43° C. The increase in temperatureis achieved using, e.g., activation by light, electromagnetic radiation,microwave radiation, radiofrequency waves, focused ultrasound, oralternating magnetic field to preferentially heat the nanoparticlesbecause of their high surface to volume ratio, and because the selectedmolecular composition of the nanoparticles preferentially absorbs morethermal energy than the surrounding normal cells. The tumor cells towhich the nanoparticles are attached are also heated. The thermal energydamages also the VLP without reducing its immunogenicity at temperatureor 41-43 degrees C. so that there is no chance of multiplication of theVLP in the body.

In one embodiment, the increase in the temperature of the nanoparticlesresults in their thermal expansion. Thermal expansion of thenanoparticles produces an ultrasonic wave that passes through the body,is captured by a receiver, the ultrasonic pulse is converted andamplified by an ultrasonic, photoacoustic, or thermoacoustic unit,imaged as a thermoacoustic signal or as nanoparticle assistedthermoacoustic signal, and converted by a computer to images, in one-,two-, or three-dimensions, of the temperature and the lesion.

In one embodiment, the photoacoustic or nanoparticle assistedthermoacoustic unit controls the thermal energy delivery unit via aprocessor to maintain the temperature of the nanoparticles at apredetermined temperature as a closed circuit once the nanoparticleshave attached to the tumor cells. An increase in the temperature towhich the nanoparticles are exposed, i.e., at the nanoparticle level,from 37° C. to 41° C.-43° C. melts the thermosensitive polymers coatingthe nanoparticles, releasing under control the conjugated VLP,medication/gene which are attached to the thermosensitive antibodycoated nanoparticles locally at the desired site. This method isparticularly effective in small tumors, i.e., tumors less than 4 mm indiameter, because the tumor stem cells are still present at the originaltumor site and can be simultaneously killed and eliminated beforemetastasis has occurred.

In one embodiment, a plurality of the antibody-coated nanoparticles areinjected into a patient's circulation with the cultured andtumor-sensitized NK cells/dendritic cells to target the tumor. It willbe appreciated that such thermal damage to tumor cells, and a NKcellular response, generates and releases relatively large quantities oflytic enzymes and other cellular contents. In the case of smallertumors, the released substances are of smaller quantities, but forlarger tumors it become necessary to remove them from the blood, byplasmaphoresis, plasma exchange etc. these substances released into apatient's circulation, may be thought of as cellular debris or detritus,to prevent an immunogenic or cytokine storm in the body.

In one embodiment, the patient receiving the inventive therapy undergoesplasmapheresis to remove, e.g., such cytokines, enzymes, dead cells,etc. from the circulation. Plasmaphoresis is a known method to removecomponents from blood plasma. Because the patient's plasma is treatedextracorporeally, then reinfused, in contrast to reinfusing onlycellular components of the patient's blood, plasmaphoresis alsobeneficially detoxifies the patient's plasma without compromising bloodvolume and with minimal or no fluid loss. This technique avoids theserious complications and side effects of simply returning the cellularcomponents of the blood to the patient. Additionally, all precautionsare observed to avoid hypotension and loss of calcium ions in theprocess of citrate anticoagulation that this procedure requires. Thepatient can be treated initially with presently available anticoagulantssuch as heparin, coumadin, etc., which can be immediately neutralizedpost-procedure. Neutralization uses standard techniques known in theart, such as calcium, etc. Hemofiltration treatment is performed withactivated carbon, treatment on non-ionic exchange resins, etc. forremoving free toxin and also toxin bound with plasma proteins, etc. asin renal dialysis methods. The process may be instituted or repeated asneeded, e.g., if the tumor reappears. The addition of Rock inhibitors orWnt inhibitors along with other therapeutic agents can reduce the everinflammatory response seen in immune therapy or an auto immune disease.

In one embodiment, to prevent a severe autoimmune response after tumorimmunotherapy, one uses extracorporeal plasmapheresis. A strong pulse oflight energy is applied to a tube containing blood cells to achieve atemperature up to 60° C. to kill immune cells containing quantum dots.The blood is then passed through a dielectrophoresis system tocharacterize and remove dead or live T-cells, sensitized killer cells,and tumor cells, prior to re-infusing the same blood or performing ablood transfusion in the patient while simultaneously administeringimmunosuppressive agents, including a biologic, to reduce the severeautoimmune response often seen after tumor immunotherapy.

The size of the nanoparticle may vary, and may vary depending on thesite of therapy and imaging as well as other factors. In one embodiment,the nanoparticle size ranges from 1 nm to 999 nm or more. In oneembodiment, the nanoparticle size ranges from 1 nm to 20 nm, which isideal for use in the eye and central nervous system to permit thenanoparticle access to the intercellular space, and also ideal for renalclearance without generating systemic side effects. Nanoparticles havinga size less than 10 nm in diameter, and not bound to a tumor, i.e.,nanoparticles that are free in the circulation, undergo rapid renalelimination from the body within a few hours of administration. Onlynanoparticles attached to the tumor cells remain in the body. Thisresults in a novel form of simultaneous local thermotherapy andvaccination.

The localized thermotherapy component of the method damages the tumorcells, thus disseminating tumor cell-associated antigens into thecirculation, generating a cellular immune response to the various tumorbiomarkers that were originally present. This dual thermotherapy andcellular response augments the effect of both immunotherapy andthermotherapy. The inventive method augments immunotherapy methods thatrelied on T-cells that had been sensitized to just a few tumor markers,or that relied only on checkpoint inhibitors to prevent the tumor cells'sequestration from T-lymphocytes. Previous methods of tumor vaccinationused intradermal or subcutaneous antigen administration, with theantigen taken up by antigen presenting cells, e.g., dendritic cells, togenerate specific killer cells only at a location remote from thespecific tumor site. The inventive method augments the previousimmunotherapy methods by combining immunotherapy to act synergisticallywith thermotherapy locally at the tumor site, providing exposure of theentire body to therapeutic agents or checkpoint inhibitors that causesimmune suppression or auto-immune disease.

In one embodiment, cultured killer cells sensitized to a tumor areadministered simultaneously with the anti-tumor antibody coatednanoparticle-conjugated VLP to attack the tumor cells by the patientst-lymphocytes etc. and remove the dead tumor cells. For example, anintradermally administered antitumor antibody-coated nanoparticle withVLP can be administered with checkpoint inhibitors such as PD-1, PD-L1,CTLA-4, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, such asFasudil, botox or Wnt inhibitors, such as niclosamide, in subsequentrounds of therapy during a postoperative period to induce an immuneresponse as needed. This embodiment decreases the likelihood of, orprevents potential recurrences of the tumor or treats a recurrence of atumor as a vaccination or therapy in recurrences. Since tumorrecurrences are generally non-sensitive to ordinary therapy, it isimportant that vaccination is done along with the checkpoint inhibitorsto be able to attack the tumor and the cell recurrences that potentiallyhave survived the previous therapy addition or Rock inhibitors or Wntinhibitors reduces excessive inflammatory disease and discourage tumorcell proliferation.

In one embodiment for use in larger tumors of a sufficient size forbiopsy, an antibody directed to the tumor lysate (TL) is used as asource of tumor-associated antigens (TAAs), and is conjugated with thenanoparticles for generating therapeutic anti-tumor immune responses.One can generate in vivo immunity against multiple TAA simultaneouslyfrom the killed or damaged tumor cells during the thermotherapy. Thisembodiment broadens the repertoire of TAA-specific T-cell clonesavailable for activation or therapy of these tumors.

In one embodiment, after an initial thermotherapy procedure, a bloodsample is obtained from the patient. This blood sample contains releasedtumor antigens that are recoverable prior to treatment by the inventivemethod using various immunoassays or methods of searching forbiomarkers. The tumor antigens are then used to generate, in vitro,additional T-cells that are sensitized to many TAA for future use in,along with VLP for vaccination, and are administered with checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.,and Rock inhibitors, such as Fasudil, etc. or Wnt inhibitors, such asniclosamide, etc. to the same patient to enhance the immune response asa vaccine or to treat potential recurrences of the same tumor.

In one embodiment, immunostimulatory oligonucleotide-loaded cationicgraphene oxide, carbon nanotube, gold/iron, iron/zinc oxide, or cadmiumsulfate nanoparticles are combined with photothermally enhancedimmunogenicity to achieve combined thermo-immune therapy. In oneembodiment, RNA oligonucleotides/graphene or graphene oxide, or longdouble stranded RNA/graphene oxide induces a controlledimmunostimulation in combination with oncogene silencing RNAi.

Nanoparticles, dendrimers, carbon nanotubes, lipid-based carriers,micelles, gold nanoshells/nanocages, PLGA, chitosan, PEI cationic lipid,and cationic polymers are useful gene therapy, gene delivery, and immunotherapy. These have the advantages of being easily prepared,biodegradable, non-immunogenic, non-toxic, and water soluble.

EXAMPLE 1

T cells and dendritic cells are obtained from a patient's blood, andgrown in culture along with a tumor or other antigen, plus nanoparticlescoated with thermosensitive polymers conjugated with antigen and VLPusing culture methods known in the art.

The nanoparticle complex is injecting them along with checkpointinhibitors and IL-2. The inventive method is applied, killing tumorcells, and increasing the response of T-cells and dendritic cell.

The patient's blood is assessed for new biomarkers from the dead cells.

The cultured T-cells and dendritic cells are harvested, along with thenanoparticle-coated antigen plus VLP or RNA or DNA phages. These arestored under appropriate conditions, and reinjected into the patientwith low dose coated nanoparticles or systemic medicaments to beadministered with checkpoint inhibitors such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor 15D11, etc. and Rock inhibitor, Fasudil or Wntinhibitor such as niclosamide as needed, e.g., semi-annually, annually,biannually, etc. as vaccination or in tumor recurrences in metastaticdisease with repetition as needed. This is followed up with counting orquantifying circulating DNA, exosomes or circulating cells to recognizepotential tumor recurrences.

EXAMPLE 2

A checkpoint inhibitors and rock inhibitors, e.g., Botox up to 50-100picograms (pg), or 100 picograms (pg) to 1 nanogram (ng) or more orfasudil from 100 picograms (pg) to 10 nanograms (ng) or 50 nanograms(ng) to 1 milligrams (mg) or Wnt inhibitors, e.g., niclosamide is addedto a thermosensitive polymer conjugated with and antibody coatedpluralities of nanoparticle for controlled release with the checkpointinhibitor using the inventive controlled thermotherapy, NATTI, torelease the medication locally at temperature of 41-43 C degree alongimmune stimulators such as VLP to treat a patient with breast,colorectal, glioblastoms, prostate, eye or skin melanomas, pancreatic,lung cancer, and/or ovarian cancer etc. A checkpoint inhibitor, such asnivolumab or PD-1L, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rockinhibitors, such as Fasudil or botox, or Wnt inhibitors, such asniclosamide or ivermectin, is combined with pluralities of antibodycoated nanoparticle assisted targeted immunotherapy for adaptive T-celltransfer to overcome the limitations of standard immunotherapy andprevent a cytokine storm. In one embodiment the Rock inhibitor Fasudilcan be taken orally at the dose of 40-80 mg as needed and niclosamide1-2 gram once or repeated in a week ivermeting also can be given orallyat a dose of 1 gram orally for a period of time during and shortly afterthermotherapy for a few days as needed.

EXAMPLE 3

Nanoparticles are conjugated with a chimeric receptor, a CD19 proteinthat is found only on B cells, along with the T-cells cultured in vitrothat expresses a chimeric antigen receptor (chimeric antigen receptor T(CAR T)-cells) to target abnormal B cells seen in leukemia along withPD-1L, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, suchas Fasudil or botox, or Wnt inhibitors, such as niclosamide. Thereappearance of new biomarkers as neoantigens in these patients can bealso treated in the postoperative period using the inventive methodrepeated therapy as vaccination along with Rock inhibitor encourage theabnormal mature cells to undergo apoptotic degeneration rather cellproliferation with abnormal genetic changes which is characterized bythe tumors occurring as a result of aging process and not a pre-existinggenetic mutation.

Plasmaphoresis is simultaneously performed or performed after treatment.

This example treats acute and chronic hematologic malignancies such asacute lymphoblastic leukemia, non-Hodgkin lymphoma, chronic lymphocyticleukemia, chronic myelogenous leukemia, etc.

EXAMPLE 4

Pluralities of antibody coated nanoparticles are conjugated withall-trans retinoic acid (ATRA) and arsenic trioxide to target leukaemiacells in acute promyelocytic leukemia and used in the inventive method.The all-trans retinoic acid is released at the site of the tumor withoutexposing the entire body to the toxic medication, simultaneously,plasmophoresis is performed to clear all toxin released in the blood,along with leukemic cells. It is appreciated that other blood cellcancers are removed in the same session.

EXAMPLE 5

In a patient with a hematologic malignancy that is resistant tochemotherapeutic agents or immune therapy, NATTI is performed with genedelivery, along with chemotherapeutic agents, to target all immune cellsinitially without subjecting the patient to systemic heavy chemotherapy,followed by bone marrow transplantation, without exposing the entirebody to systemic chemotherapy.

EXAMPLE 6

Pluralities of antibody coated nanoparticles are conjugated with RNAthat contains an aptamer, ribosomes, and siRNA in a thermosensitivepolymer and administered to using NATTI to target specific tumor cells.

EXAMPLE 7

The microenvironment of the cancer cell is modified by deliveringmedicaments that block the uptake of exosomal signals and prevent theuptake of ECV. Such medications include choloroquine, heparin,cytochalasin D, and ethyl-isopropyl amiloride are conjugated withpolymeric coating and conjugated with antibody coated nanoparticlesadministered to the patient and released with NATTI. These medicationsare approved for patient use. The medicaments are provided using NATTIin conjunction with chemotherapeutic agents and rock inhibitors.

EXAMPLE 8

The inventive method provides nanoparticle assisted localizedimmunothermotherapy and thermotherapy for delivery of customizedvaccines with or without VLP to target core mutations in a patient. Theimmune cells or T-cells that can attack those core mutations areidentified via a cancer biomarker. The immune cells or T-cells are thencultured with the nanoparticles coated with thermosensitive particlesand VLP and IL-2. The antibody coated nanoparticles with checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.,and Rock inhibitor, such as Fasudil or Botox etc., or Wnt inhibitor,such as niclosamide, etc., are injected into the patient, controllablyheated using a thermal energy source, and imaged, for specific patient,or those with metastatic disease or recurrences as immunotherapy, suchas in breast cancer, prostate cancer, glioblastoma, lung cancer,melanoma, ovarian cancer, pancreatic cancer, intestinal or colon cancer,etc.

EXAMPLE 9

Antibody coated nanoparticles are conjugated with RNA phage VLP, whichis generally stable. VLPs of the related RNA phage PP7 are crosslinkedwith inter-subunit disulfide bonds, rendering them significantly morestable. They exhibit high immunogenicity. Such nanoparticles complementthe inventive NATTI technology and can be employed in anti-cancer andantibacterial treatment. Lytic phages attach to receptors on thebacterial surface, inject their genetic material through the bacterialmembrane, and overtake the bacterium's transcription and translationmachinery to synthesize new phages. The application of thermotherapydamages all VLP, phages, virocides or foreign protein and eliminatetheir future growth and potential adverse reactions.

EXAMPLE 10

To prevent a severe autoimmune response after tumor immunotherapy,before extracorporeal plasmapheresis, one uses the nanoparticle assistedthermotherapy and imaging system to apply heavy thermal energy to a tubecontaining blood cells and to achieve a temperature as high as 60° C. tokill the sensitized immune cells containing nanoparticles. Blood is thenpassed through a dielectrophoresis system to characterize and removedead or live T-cells, sensitized killer cells, and dead tumor cellsprior to re-infusing blood in the patient while simultaneouslyadministering immunosuppressive agents and Rock inhibitors, includingbiologics. This reduces the severe autoimmune response often seen aftertumor immunotherapy.

The embodiments shown and described in the specification are onlyspecific embodiments of the inventor who is skilled in the art and arenot limiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

What is claimed is:
 1. A cancer therapeutic method comprising administering to a patient having an early stage tumor a combination of thermotherapy and immunotherapy, where thermotherapy comprises systemically administering a plurality of tumor-antibody-coated nanoparticles coated with a thermosensitive polymer, the thermotherapy further comprises heating the tumor-antibody-coated nanoparticles using an energy source at the site of the tumor so as to damage one or more tumor cell membranes and release antigenic material in vivo that activates and stimulates an immunogenic response of the patient at the site of the tumor; and immunotherapy comprises systemically administering the patient's natural killer (NK) cells/dendritic cells pre-sensitized in vitro to the tumor.
 2. The method of claim 1 where the step of heating the tumor-antibody-coated nanoparticles using the energy source comprises controllably increasing the temperature to which the tumor-antibody-coated nanoparticles are exposed from 37° C. to between 41° C. and 43° C. for a predetermined time period resulting in melting the thermosensitive polymer coating the tumor-antibody-coated nanoparticles, and releasing additional tumor antigens in the circulation of the patient from the thermally damaged tumor cells; the method further comprising the steps of: obtaining from the blood of the patient, the tumor antigens to build a new potent vaccine against many additional tumor specific antigens, the vaccine combined with tumor-antibody-coated nanoparticles conjugated with checkpoint inhibitors, and Rock inhibitors or Wnt inhibitors; and administering the vaccine with the tumor-antibody-coated nanoparticles conjugated with viral-like particles (VLP) while simultaneously releasing the conjugated checkpoint inhibitors, and Rock inhibitors or Wnt inhibitors, from the tumor-antibody-coated antibody coated nanoparticles to prevent new or old tumor cells or metastatic cells from being disguised from the T-lymphocytes or the patient's natural killer (NK) cells, thereby providing a vaccine for treatment of potential recurrences of the same tumor to the patient and enhancing the immune response at the specific location of one or more metastatic lesions, circulating tumor cells, or sessile tumor cells.
 3. The method of claim 2 where the checkpoint inhibitors conjugated with the tumor-antibody-coated nanoparticles are selected from the group consisting of PD-1, PD-L1, CTLA-4, jagged 1 inhibitor 15D11, and combinations thereof.
 4. The method of claim 2 where the tumor-antibody-coated nanoparticles are conjugated with Rock inhibitors, the Rock inhibitors selected from the group consisting of Fasudil, exoenzyme, Y27632, Botox, and combinations thereof.
 5. The method of claim 2 where the tumor-antibody-coated nanoparticles are conjugated with Wnt inhibitors, the Wnt inhibitors selected from the group consisting of niclosamide, ivermectin, and combinations thereof.
 6. The method of claim 2 further comprising the step of: repeating administration of the vaccine together with checkpoint inhibitors and Rock inhibitors once or twice a year, thereby reducing an auto-immune reaction.
 7. The method of claim 1 where the tumor-antibody-coated nanoparticles are conjugated with a medication, and where thermotherapy includes exposing the tumor-antibody-coated nanoparticles and medication to a light pulse at a frequency in the range of 20 Hz to 60 Hz to decrease proliferation of the tumor cells.
 8. The method of claim 1 where immunotherapy is administered at intervals to the patient after the initial therapy acting as a booster to the original immunotherapy and reduce or prevent tumor recurrences at a same or different site.
 9. The method of claim 1 where immunotherapy further comprises obtaining NK cells/dendritic cells grown in culture under light pulses with a tumor biomarker from blood or a tumor biopsy specimen containing tumor lysate, killed circulating tumor cells (ct cell), and tumor extracellular vesicles (ECV).
 10. The method of claim 1 where thermotherapy includes exposing the tumor-antibody-coated nanoparticles to a light pulse at a frequency in the range of 20 Hz-60 Hz to decrease proliferation of the tumor cell.
 11. The method of claim 1 where the step of heating the tumor-antibody-coated nanoparticles using the energy source comprises using a thermoacoustic unit to control a thermal energy delivery unit using a processor to maintain the tumor-antibody-coated nanoparticles at a predetermined temperature as a closed circuit once the tumor-antibody-coated nanoparticles have attached to the tumor cells, then controllably increasing the temperature to which the tumor-antibody-coated nanoparticles are exposed from 37° C. to 41° C.-43° C. for a predetermined desired time period resulting in melting the thermosensitive polymer coating the tumor-antibody-coated nanoparticles, and releasing under control a medication or gene, which are attached to the thermosensitive tumor-antibody-coated nanoparticles locally at the desired site.
 12. The method of claim 1 where the anti-tumor antibody is specific for at least one tumor biomarker in the patient's blood.
 13. The method of claim 1 where the polymer contains an inhibitory gene(s) and a CRISPR/cas9 complex to stimulate or modify tumor genes at the desired site upon release from the polymer at a desired temperature that is obtained by incremental increase from 37° C. to 43° C. permitting precision nanoparticle assisted thermotherapy and imaging (NATTI) to release the gene(s) and optional medicament(s) and/or checkpoint inhibitor(s) from the nanoparticles.
 14. The method of claim 1 further comprising conjugating the nanoparticles with a chimeric receptor on B cells and with T-cells cultured in vitro and expressing an antigen for the chimeric receptor to target abnormal B cells seen in leukemia.
 15. The method of claim 1 further comprising performing precision nanoparticle assisted thermotherapy and generating a photoacoustic image of the cells to which the nanoparticles bind at an early cellular stage having a size between one and two millimeters in diameter not easily detectable by radiographic imaging.
 16. A method of simultaneous localized thermotherapy and vaccination, the method comprising: administering a plurality of nanoparticles to a patient in need thereof, the administered nanoparticles having a size less than 10 nm in diameter and coated with an antitumor antibody, the patient provided nanoparticle assisted thermotherapy, the unbound nanoparticles undergoing renal elimination from the body within a plurality of hours of administration, and the tumor-bound nanoparticles remaining in the patient, wherein the nanoparticle assisted thermotherapy comprises heating the tumor-bound nanoparticles using an energy source at the site of the tumor so as to damage one or more tumor w cell membranes and release antigenic material in vivo that activates and stimulates an immunogenic response by the natural killer (NK) cells/dendritic cells of the patient.
 17. A therapeutic method to treat a pathology implicating a cellular protein(s), the method comprising administering to a patient having a cellular pathology a combination of thermotherapy, and immunotherapy, with gene delivery, where: thermotherapy comprises systemically administering antibody-coated nanoparticles coated with a thermosensitive polymer and conjugated with one or more genes for gene delivery to a diseased cell, the thermotherapy further comprises heating the antibody-coated nanoparticles using an energy source to release the one or more genes at the site of the diseased cell to effect gene therapy; immunotherapy comprises systemically administering the patient's natural killer (NK) it) cells/dendritic cells pre-sensitized in vitro to the cellular protein(s); and wherein the gene therapy comprises use of a CRISPR/cas9 and/or CRISPRi to replace or modify at least one genetic component in the diseased cell.
 18. The method of claim 17, further comprising performing plasmaphoresis on the patient post-therapy to purify a patient's blood from toxins and cellular components generated by the therapy.
 19. The method of claim 17, further comprising: applying light energy to a tube containing the patient's blood cells post-therapy to achieve a temperature up to 60° C. to kill immune cells containing nanoparticles; passing the pulsed blood cells through a dielectrophoresis system to characterize and remove dead or live T-cells, sensitized killer cells, and tumor cells; and re-infusing the dielectrophoresis treated blood in the patient while simultaneously administering immunosuppressive agents, thus reducing the likelihood of a severe post-therapy autoimmune response in the patient. 